Biological fertilizer compositions comprising sludge

ABSTRACT

The present invention provides biological fertilizer compositions that comprise yeast cells and sludge. The yeast cells of the invention have an enhanced ability to fix atmospheric nitrogen, decompose phosphorus minerals and compounds, decompose potassium minerals and compounds, decompose complex carbon compounds, overproduce growth factors, overproduce ATP, decompose undesirable chemicals, suppress growth of pathogenic microorganisms, or reduce undesirable odor. The biological fertilizer composition of the invention can replace mineral fertilizers in supplying nitrogen, phosphorus, and potassium to crop plants. Methods of manufacturing biological fertilizer compositions, and methods of uses are also encompassed.

1. FIELD OF THE INVENTION

[0001] The invention relates to biological fertilizers that compriseyeasts and an organic substrate. The yeasts in the compositions of theinvention have been stimulated to perform a variety of functionsincluding the conversion of the organic materials into non-hazardousplant nutrients. The invention also relates to methods for manufacturingbiological fertilizers, and methods for using the biological fertilizersto increase crop yields.

2. BACKGROUND OF THE INVENTION

[0002] Use of fertilizer is essential in supporting the growth of highyield crops. Of the basic nutrients that plants need for healthy growth,large amounts of nitrogen (taken up as NO₃ ⁻ or NH₄ ⁺), phosphorus(taken up as H₂PO₄ ⁻), and potassium (taken up as K⁺) nutrients arerequired by most crops on most soils (Wichmann, W., et al., IFA WorldFertilizer Use Manual). Such large amounts of nitrogen, phosphorus, andpotassium nutrients are supplied mainly in the form of mineralfertilizers, either processed natural minerals or manufactured chemicals(K. F. Isherwood, 1998, Mineral Fertilizer Use and the Environment,United Nations Environmental Programme Technical Report No. 26).

[0003] Despite the importance of mineral fertilizers in providingmankind with abundant agricultural products, the harm done to theenvironment has been recognized in recent years. Mineral fertilizers mayincurred damages to soils. For example, most nitrogen fertilizers mayacidify soils, thereby adversely affecting the growth of plants andother soil organisms. Extensive use of chemical nitrogen fertilizers mayalso inhibit the activity of natural nitrogen fixing microorganisms,thereby decreasing the natural fertility of soils. The long term use ofmineral fertilizers may also cause severe environmental pollution. Forexample, the loss of nitrogen and phosphate fertilizers due to leachingand soil erosion has led to contamination of soil and ground water, andeutrophication of surface water. Cleaning up polluted soil and water hasbeen a complicated and difficult task. The cost for such a task is alsoastronomical.

[0004] In search for a solution to the problem, some are going back toorganic fertilizers, such as manure (Wichmann, W., et al., IFA WorldFertilizer Use Manual). The use of manure as fertilizer dates to thebeginnings of agriculture. Large amounts of manure are produced bylivestock. For example, in the United States, farms (including confinedanimal feeding operations) generate more than 136 million metric tons(dry weight basis) of waste products annually. Manure has value inmaintaining and improving soil because of the plant nutrients, humus,and organic substances contained in it. Studies have shown that a highpercentage of the nitrogen, phosphorus, and potassium fed to dairycattle are excreted in manure.

[0005] As manure must be managed carefully in order to derive the mostbenefit from it, some farmers may be unwilling to expend the necessarytime and effort. Manure must be carefully stored to minimize loss ofnutrients. It must be applied to the right kind of crop at the propertime. In general, manure does not provide all the plant nutrients neededand very large amount of organic fertilizers have to be applied to soil.Thus, there is a tendency to discount the value of manure as fertilizer.Manure may also contain undesirable chemicals, such as antibiotics andhormones. Only in underdeveloped countries, where artificial fertilizermay be costly or unavailable and where labor is relatively cheap, manureis attractive as a fertilizer.

[0006] Furthermore, manure may contain significant levels of nitrogenand phosphorous which threaten water resources if not managed correctly.If not stored or disposed of properly, it can pose health andenvironmental threats. For example, it can cause air pollution, i.e.,odor and dust; and contamination of surface and ground water with excessnutrients, organic matter, salts, and pathogens. For example, manurecontains pathogenic microorganisms, such as Escherichia coli Salmonellaspp., Shigella spp., and Campylobacter jejuni.

[0007] Biological fertilizers utilizing microorganisms have beenproposed as alternatives to mineral fertilizers. Naturally occurringnitrogen fixing microorganisms including bacteria, such as Rhizobium,Azotobacter, and Azospirillum, (See for example, U. S. Pat. No.5,071,462) and fungi, such as Aspergillus flavus-oryzae, (See, forexample, U. S. Pat. No. 4,670,037) have been utilized in biologicalfertilizers. Naturally occurring microorganisms capable of solubilizingphosphate rock ore or other insoluble phosphates into soluble phosphateshave also been utilized in biological fertilizers either separately(e.g., U. S. Pat. No. 5,912,398) or in combination with nitrogen fixingmicroorganisms (e.g., U. S. Pat. No. 5,484,464). Genetically modifiedbacterial strains have also been developed and utilized in biologicalfertilizers. An approach based on recombinant DNA techniques has beendeveloped to create more effective nitrogen fixing, phosphorusdecomposing, and potassium decomposing bacterial strains for use in abiological fertilizer, see, for example, U.S. Pat. No.5,578,486; PCTpublication WO 95/09814; Chinese patent publication: CN 1081662A; CN1082016A; CN 1082017A; CN 1103060A; and CN 1 109595A.

[0008] However, the biological fertilizers that are based on naturallyoccurring microorganisms are generally not efficient enough toeffectively replace mineral fertilizers. It is therefore important todevelop more advanced biological fertilizers that can replace mineralfertilizers in supplying nitrogen, phosphorus, and potassium to cropsfor producing high quality agricultural products while avoiding theproblems associated with mineral fertilizers.

[0009] The present invention provides a biological fertilizer based onnon-recombinant yeasts, which can replace mineral fertilizers andprovide an effective and environmentally-friendly method of usingcertain organic materials.

[0010] Citation of documents herein is not intended as an admission thatany of the documents cited herein is pertinent prior art, or anadmission that the cited documents are considered material to thepatentability of the claims of the present application. All statementsas to the date or representations as to the contents of these documentsare based on the information available to the applicant and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

3. SUMMARY OF THE INVENTION

[0011] The present invention relates to biological fertilizercompositions. The biological fertilizer compositions of the inventioncomprises up to nine different yeast cell components, sludge, andoptionally an inorganic substrate component. In particular, the yeastcell components of the composition are each capable of at least one ofthe following ten functions, namely, fixing atmospheric nitrogen,decomposing insoluble phosphorus or potassium minerals, maintaining abalance of phosphorus compounds, decomposing complex carbon-containingmaterials or compounds, overproducing growth factors, overproducing ATP,suppression of growth of pathogenic microorganisms, breakdown ofundesirable chemicals, and reducing the odor of organic matters,respectively. The yeast cell components of the invention can be used asan additive which is mixed with sludge to form a biological fertilizer.

[0012] In one embodiment, the biological fertilizer compositions of theinvention are produced by mixing sludge with at least seven and up tonine yeast cell components, wherein the cells of six yeast cellcomponents perform the basic functions of fixing atmospheric nitrogen,decomposing phosphorus-containing minerals or maintaining a balance ofphosphorus compounds, decomposing potassium-containing minerals,decomposing complex carbon-containing materials or compounds,overproducing growth factors, and overproducing ATP, and wherein thecells of the other component(s) perform the supplementary functions ofsuppressing growth of pathogenic microorganisms, decomposing undesirablechemicals, and reducing the odor of the organic substrate in thefertilizer composition.

[0013] In preferred embodiments, the present invention uses yeasts thatare commercially available and/or accessible to the public, such as butnot limited to Saccharomyces cerevisiae. Generally, the yeast cellcomponents of the invention are produced by culturing the pluralities ofyeast cells under activation conditions such that the abilities of thepluralities of cells to perform the functions are activated or enhanced.Accordingly, in another embodiment, the invention encompasses methods ofactivating or enhancing the abilities of yeast cells to perform one ofthe ten functions. The invention also relates to methods formanufacturing the fertilizer comprising mixing sludge with the yeastcells of the present invention, followed by drying and packing the finalproduct.

[0014] The invention further relates to methods for using the fertilizercompositions of the present invention. The biological fertilizercompositions of the present invention are used to support and enhancethe growth and maturation of a wide variety of plants.

4. BRIEF DESCRIPTION OF FIGURES

[0015]FIG. 1 Activation of yeast cells. 1 yeast cell culture; 2container; 3 electromagnetic field source.

[0016]FIG. 2. Formation of symbiosis-like relationships among strains ofyeasts. 4 electromagnetic field source for nitrogen-fixing yeasts; 5electromagnetic field source for P-decomposing yeasts; 6 electromagneticfield source for K-decomposing yeasts; 7 electromagnetic field sourcefor C-decomposing yeasts; 8 yeast cell culture; 9 container.

[0017]FIG. 3. Adaptation of yeast cells to a soil type. 10 electrode; 11container; 12 electrode; 13 yeast cell culture; 14 electromagnetic fieldsource; 15 temperature controller.

[0018]FIG. 4. Organic substrate grinding process. 16 organic rawmaterial; 17 crusher; 18 grinder; 19 organic substrate in powder form.

[0019]FIG. 5. Inorganic substrate grinding process. 20 inorganic rawmaterial; 21 crusher; 22 grinder; 23 inorganic substrate in powder form.

[0020]FIG. 6. Yeast fermentation process. 24 activated yeast cells; 25tank for culturing yeast cells, starch: water (35° C.)=1:2.5,semi-aerobic fermentation at 28 to 30° C.; 26 harvested culture.

[0021]FIG. 7. Mixing organic and inorganic raw materials. 27 inorganicmaterials; 28 starch; 29 organic materials; 30 mixer; 31 mixture; 32mixture to be transported to fertilizer production stage.

[0022]FIG. 8. Mixing yeast cells. 33 nitrogen-fixing yeasts 34P-decomposing yeasts; 37 K-decomposing yeasts; 55 C-decomposingmicrobes; 35 ATP-producing yeasts; 36 GF-producing yeasts; 52pathogen-suppressing yeasts; 53 yeasts that decompose undesirablechemicals; 54 deodorizing yeasts; 38 mixture of yeasts; 56 mixture to betransported to fertilizer production stage.

[0023]FIG. 9. Fertilizer production process. 39 mixture of yeasts; 40mixture of organic and inorganic materials; 41 granulizer; 42 fertilizergranules.

[0024]FIG. 10. Drying process. 43 fertilizer granules; 44 first dryer;45 second dryer; 46 dried fertilizer.

[0025]FIG. 11. Cooling and packaging process. 47 dried fertilizer; 48cooler; 49 separator; 50 bulk bag filler; 51 final product.

5. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides biological fertilizer compositionsthat comprise yeast cells and sludge. The present invention alsoprovides methods for manufacturing the biological fertilizercompositions as well as methods for using the biological fertilizercompositions.

[0027] The biological fertilizer compositions of the invention canreplace chemical/mineral fertilizers in supplying nitrogen (N),phosphorus (P), and potassium (K) to plants, especially crop plants. Theinclusion of sludge in the biological fertilizer compositions of theinvention provide an environmentally acceptable and economic method forrecycling sludge.

[0028] According to the invention, the biological fertilizercompositions comprise poultry manure and a plurality of yeast cellcomponents. Each yeast cell component is a population of yeast cellswhich comprises a plurality of yeast cells that are capable ofperforming a desired function. The yeast cell components of theinvention can provide the following six basic functions: (1) fixation ofatmospheric nitrogen; (2) decomposition of phosphorus minerals orcompounds, or maintaining a balance of phosphorus compounds; (3)decomposition of potassium minerals or compounds; (4) decomposition ofcomplex or high molecular weight carbon materials or compounds; (5)overproduction of growth factors; and (6) overproduction of ATP. Theyeast cell components of the invention can provide the followingsupplementary functions: (7) suppression of growth of pathogens, (8)degradation of undesirable chemicals, or (9) reducing the odor oforganic materials.

[0029] In one embodiment, a biological fertilizer composition of theinvention comprises (I) poultry manure; (II) at least one of thefollowing yeast cell component: (a) a first yeast cell componentcomprising a first plurality of yeast cells that fix nitrogen; (b) asecond yeast cell component comprising a second plurality of yeast cellsthat decompose phosphorus compounds; or (c) a third yeast cell componentcomprising a third plurality of yeast cells that decompose potassiumcompounds; and (III) at least one of the following: (d) a fourth yeastcell component comprising a fourth plurality of yeast cells thatsuppress the growth of pathogenic microorganisms; (e) a fifth yeast cellcomponent comprising a fifth plurality of yeast cells that degradeantibiotics; or (f) a sixth yeast cell component comprising a sixthplurality of yeast cells that reduce the odor of the biologicalfertilizer composition. Thus, a biological fertilizer composition of theinvention comprises at least two yeast cell components, one providingone of the three listed basic functions and one providing asupplementary function. In another embodiment, the biological fertilizercomposition as described above further comprises at least one of thefollowing: (g) a seventh yeast cell component comprising a seventhplurality of yeast cells that convert complex carbon compounds to simplecarbohydrates; (h) an eighth yeast cell component comprising an eighthplurality of yeast cells that overproduce growth factors; or (i) a ninthyeast cell component comprising a ninth plurality of yeast cells thatoverproduce adenosine triphosphate. In preferred embodiments, thebiological fertilizer compositions of the invention comprises yeast cellcomponents that provide all six basic functions, plus at least one ofthe supplementary functions. Thus, the preferred biological fertilizercompositions comprise seven, eight or nine different yeast cellcomponents.

[0030] The pluralities of the yeast cells of the invention can be addedto sludge or existing organic fertilizers to improve their performance.

[0031] The sludge in the fertilizer compositions provides a source ofnitrogen, hosphorus and potassium. Optionally, the fertilizercompositions may include an inorganic component comprising mineralswhich provides an additional source of phosphorous and/or potassium, andother minerals such as but not limited to calcium, magnesium, andsulfur; and micronutrients, such as but not limited to boron, copper,iron, manganese, molybdenum, and zinc.

[0032] The biological fertilizer compositions of the present inventionhave many advantages over mineral fertilizers and organic fertilizers.Because the biological fertilizer of the present invention utilizemetabolic activities of living yeasts to convert raw materials, such asatmospheric nitrogen, and phosphorus and potassium compounds in thesubstrate component, into plant nutrients, the conversion and release ofsuch nutrients by the yeast cells is regulated in part by the nutrientcontent of the soil. The nutrient content of the soil in turn depends inpart on both the environment and the changing needs of plants.Therefore, the release of plant nutrients by the biological fertilizercompositions is adaptable to the soil condition and can be sustainedover a period of time.

[0033] In addition to supplying nutrients to plants, the biologicalfertilizer compositions of the invention provide up to threesupplementary functions that mitigate some of the undesirable propertiesof sludge that tend to restrict their use as organic fertilizers. Thepresence of pathogenic bacteria in sludge poses a health risk to humansand livestock. The biological fertilizer compositions can include acomponent of yeast cells that can suppress the proliferation ofpathogenic bacteria, thereby reducing the risk of infection, andcircumventing the need to use chemicals in controlling the spread ofsuch pathogens. Another yeast cell component that can be included in thecomposition is capable of reducing the odor of sludge, thus making itsinclusion in a fertilizer more acceptable. Yet another yeast cellcomponent can be included to degrade undesirable chemicals, such asantibiotic feed additives, which are found in sludge. Thesesupplementary functions generally lessen the adverse impact on theenvironment of using sludge in a fertilizer. The yeast cell componentsthat provide the supplementary functions can each be separately includedwith the other six yeast cell components that provide the basicfunctions, or in combination with each other and the other sixcomponents to provide the desired assortment of supplementary functions.

[0034] While the following terms are believed to have well-definedmeanings in the art, the following are set forth to facilitateexplanation of the invention.

[0035] As used herein, the term “nitrogen fixation” or “fixation ofatmospheric nitrogen” encompasses biological processes in whichmolecular nitrogen or nitrogen in the atmosphere is converted into oneor more nitrogenous (N) compounds, including but not limited to,ammonia, ammonium salts, urea, nitrites, and nitrates.

[0036] As used herein, the phrase “decomposition of phosphorus mineralsor compounds” refers to biological processes which convert phosphorus(P) compounds, such as but not limited to those water-insolublephosphorus compounds present in minerals, such as phosphate rock, intoone or more different phosphorus compound(s) which are biologicallyavailable or more readily assimilable, i.e., usable for survival and/orgrowth, by plants and other yeasts. For example, the resultingphosphorus compounds may be more soluble in water or weak acid, and canthus be taken up by the roots of plants. Non-limiting examples ofbiologically available or assimilable phosphorus compounds includevarious classes of phosphates such as H₂PO₄ ⁻ and HPO₄ ²⁻.

[0037] As used herein, the phrase “maintenance of a balance ofphosphorus compounds” refers to biological processes which convertbiologically unavailable or water-insoluble phosphorus compounds intoone or more different phosphorus compound(s) which are more biologicallyavailable or soluble in water, wherein the processes are sensitive toexcess or the lack of phosphorus (P) compounds in the local environment.The conversion process is downregulated when the level of P compound ishigh (i.e., greater than about 180 ppm) and upregulated when level of Pcompound is low (i.e., greater than about 60 ppm)

[0038] As used herein, the phrase “decomposition of potassium mineralsor compounds” refers to biological processes which convert potassium (K)compounds, such as but not limited to those water-insoluble potassiumcompounds present in potassium-containing minerals and materials, intoone or more different potassium compound(s) which can be biologicallyavailable or more readily assimilable by plants and other yeasts. Forexample, the resulting potassium compounds may be more soluble in water,and can thus be taken up by the roots of plants.

[0039] As used herein, the phrase “decomposition of complex or highmolecular weight carbon minerals, materials or compounds” refers to thebiological conversion of a complex organic or inorganic carbon molecule(e.g. complex carbohydrates like cellulose and lignin into one or morecarbon compound(s) which are of a lower molecular weight (e.g., simplecarbohydrates) and can be readily used for survival and/or growth byplants and yeasts. This process includes those reactions where longchains of carbon atoms in a polymeric carbon compound are cleaved.

[0040] As used herein, the term “growth factors” refers to moleculescommonly required for the growth of yeasts, including but not limited tovitamins, in particular, vitamin B complexes, e.g., vitamin B-1,riboflavin (vitamin B-2), vitamin B-12, niacin (B-3), pyridoxine (B-6),pantothenic acid (B-5); folic acid; biotin; para-aminobenzoic acid;choline; and inositol.

[0041] For the purpose of this invention, the above-described fivefunctions together with the overproduction of growth factors and ATP arereferred to as the basic functions.

[0042] As used herein, the phrase “suppressing the growth of pathogens”refers to a decrease or lack of increase in the number of pathogenicmicroorganisms present in a sample of sludge over a period of time, as aresult of the presence of the yeast cells of the invention in thesample. It is to be understood that in the absence of the yeast cells,the number of pathogens in the sample would increase naturally. Manysuch microorganisms cause diseases in humans and animals, and mayinclude bacteria such as Escherichia species, Salmonella species,Shigella species, Mycobacterium species, Staphylococcus species,Bacillus species, Streptococcus and Diplococcus species.

[0043] As used herein, the phrase “degradation of undesirable chemicals”refers to biological or biochemical processes which result in theconversion of chemical compounds that are undesirable in a fertilizer toan inactive form, such as the breakdowvn of such compounds into lowermolecular weight compounds. Antibiotics are commonly present in organicmaterials and such compounds are not desired in a fertilizer because ofthe potential risk of ingestion by humans, for example, by eatingvegetables grown using a fertilizer comprising contaminated organicmaterial, and the possible spread of antibiotic resistance in theenvironment. Many antibiotics are added to animal feed to protectvarious farm animals, such as chicken, turkey, and swine, from bacterialand parasitic diseases, and to promote growth. A significant amount ofantibiotic feed additive is excreted by the animals, and thusaccumulates in manure and sludge. Many kinds of antibiotics have beenused in animal operations, such as but not limited to aminoglycosides,tetracyclines, beta-lactams, glycopeptides, and macrolides. Examples ofantibiotics approved for use in farms in United States include but arenot limited to, bacitracin methylene disalicylate, bacitracin zinc,bambermycins, oxytetracycline, chlortetracycline, penicillin,tylosin/sulfamethazine, roxarsone, nitrasone, monensin, lasalocid,carbodox, tiamulin, hygromycin B, nystatin, novobiocin,sulfadimethoxine, ormetroprim, lincomycin, fenbendazole, andvirginiamycin. The presence and quantity of such antibiotics in acomposition can be determined by any methods known in the art, forexample, high performance liquid chromatography (HPLC).

[0044] As used herein, the phrase “reducing the odor of organicmaterials” refers to a process which results in a lower concentration ofone or more odorous compounds in sludge. Odorous compounds, such as butnot limited to hydrogen sulfide, ammonia, indole, skatole (i.e,3-methyl-1H-indole), p-cresol, and organic acids, are known tocontribute to the malodorous quality of manure. The concentration ofsuch malodorous compounds in sludge or in a sample of air in contactwith the manure can be determined by any method well known in the art,including but not limited to gas chromatography. Odor is a perception ofsmell by an organism with olfactory organs. A reduction of the intensityof the odor associated with sludge can be determined subjectively.Various methods and techniques are known to measure the intensity of anodor. One subjective measurement of odor intensity is to measure thedilution necessary so that the odor is imperceptible or doubtful to ahuman or animal test panel. Alternatively, a recognition threshold mayalso be used which is a higher concentration at which the character ofthe odor is recognized. Any methods and techniques for objectively orsubjectively determine the intensity of an odor can be used to monitorthe performance of the compositions and methods of the invention.

[0045] For the purpose of this invention, the suppression of growth ofpathogens, degradation of undesirable chemicals, and reduction of odorof organic materials are referred to as the supplementary functions oractivities.

[0046] The inventor discovered that, under various culture conditions,yeasts can be induced to exhibit seven different basic functions andthree supplementary functions. The culture condition determines theactivity which is activated or enhanced in the cultured yeasts. Thespecific culture conditions for each of the ten functions are describedin details in sections 5.1 to 5.10 respectively.

[0047] According to the invention, a yeast cell component of thebiological fertilizer composition is produced by culturing a pluralityof yeast cells in an appropriate culture medium in the presence of analternating electromagnetic field or multiple alternatingelectromagnetic fields in series over a period of time. The culturingprocess allows yeast spores to germinate, yeast cells to grow anddivide, and can be performed as a batch process or a continuous process.As used herein, the terms “alternating electromagnetic field”,“electromagentic field” or “EM field” are synonymous. An electromagneticfield useful in the invention can be generated by various means wellknown in the art. A schematic illustration of exemplary setups aredepicted respectively in FIG. 1. An electromagnetic field of a desiredfrequency and a desired field strength is generated by anelectromagnetic wave source (3) which comprises one or more signalgenerators that are capable of generating electromagnetic waves,preferably sinusoidal waves, and preferably in the frequency range of 30MHz-3000 MHz. Such signal generators are well known in the art. Signalgenerators capable of generating signal with a narrower frequency rangecan also be used. If desirable, a signal amplifier can also be used toincrease the output signal, and thus the field strength.

[0048] The electromagnetic field can be applied to the culture by avariety of means including placing the yeast cells in close proximity toa signal emitter connected to a source of electromagnetic waves. In oneembodiment, the electromagnetic field is applied by signal emitters inthe form of electrodes that are submerged in a culture of yeast cells(1). In a preferred embodiment, one of the electrodes is a metal plate,and the other electrode comprises a plurality of wires configured insidethe container (2) so that the energy of the electromagnetic field can beevenly distributed in the culture. The number of electrode wires useddepends on both the volume of the culture and the diameter of the wire.For example, for a culture having a volume of 5000 ml, one electrodewire having a diameter of between 0.1 to 1.2 mm can be used for each 100ml of culture; for a culture having a volume greater than 1000 1, oneelectrode wire having a diameter of between 3 to 30 mm can be used foreach 1000 1 of culture.

[0049] In preferred embodiments, yeasts of the genera of Saccharomyces,Schizosaccharomyces, Sporobolomyces, Torulopsis, Trichosporon,Wickerhamia, Ashbya, Blastomyces, Candida, Citeromyces, Crebrothecium,Cryptococcus, Debaryomyces, Enclomycopsis; Geotrichum, Hansenula,Kloeckera, Lipomyces, Pichia, Rhodosporidium, and Rhodotorula can beused in the invention.

[0050] Non-limiting examples of yeast strains include Saccharomycescerevisiae Hansen, ACCC2034, ACCC2035, ACCC2036, ACCC2037, ACCC2038,ACCC2039, ACCC2040, ACCC2041, ACCC2042, AS2.1, AS2.4, AS2.11, AS2.14,AS2.16, AS2.56, AS2.69, AS2.70, AS2.93, AS2.98, AS2.101, AS2.109,AS2.110, AS2.112, AS2.139, AS2.173, AS2.174, AS2.182, AS2.196, AS2.242,AS2.336, AS2.346, AS2.369, AS2.374, AS2.375, AS2.379, AS2.380, AS2.382,AS2.390, AS2.393, AS2.395, AS2.396, AS2.397, AS2.398, AS2.399, AS2,400,AS2.406, AS2.408, AS2.409, AS2.413, AS2.414, AS2.415, AS2.416, AS2.422,AS2.423, AS2.430, AS2.431, AS2.432, AS2.451, AS2.452, AS2.453, AS2.458,AS2.460, AS2.463, AS2.467, AS2.486, AS2.501, AS2.502, AS2.503, AS2.504,AS2.516, AS2.535, AS2.536, AS2.558, AS2.560, AS2.561, AS2.562, AS2.576,AS2.593, AS2.594, AS2.614, AS2.620, AS2.628, AS2.631, AS2.666, AS2.982,AS2.1190, AS2.1364, AS2.1396, IFFI 1001, IFFI 1002, IFFI 1005, IFFI1006, IFFI 1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI 1021, IFFI 1027,IFFI 1037, IFFJ 1042, IFFI 1043, IFFI 1045, IFFI 1048, IFFI 1049, IFFI1050, IFFI 1052, IFFI 1059, IFFI 1060, IFFI 1063, IFFI 1202, IFFI 1203,IFFI 1206, IFFI 1209, IFFI 1210, IFFI 1211, IFFI 1212, IFFI 1213, IFFI1215, IFFI 1220, IFFI 1221, IFFI 1224, IFFI 1247, IFFI 1248, IFFI 1251,IFFI 1270, IFFI 1277, IFFI 1287, IFFI 1289, IFFI 1290, IFFI 1291, IFFI1291, IFFI 1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 1301, IFFI 1302,IFFI 1307, IFFI 1308, IFFI 1309, IFFI 1310, IFFI 1311, IFFI 1331, IFFI1335, IFFI 1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI 1340, IFFI 1345,IFFI 1348, IFFI 1396, IFFI 1397, IFFI 1399, IFFI 1411, IFFI 1413;Saccharomyces cerevisiae Hansen Var. ellipsoideus (Hansen) Dekker,ACCC2043, AS2.2, AS2.3, AS2.8, AS2.53, AS2.163, AS2.168, AS2.483,AS2.541, AS2.559, AS2.606, AS2.607, AS2.611, AS2.612; Saccharomyceschevalieri Guillermond, AS2.131, AS2.213; Saccharomyces delbrueckii,AS2.285; Saccharomyces delbrueckii Lindner var. mongolicus Lodder et vanRij, AS2.209, AS2.1157; Saccharomyces exiguus Hansen, AS2.349, AS2.1158;Saccharomyces fermentati (Saito) Lodder et van Rij, AS2.286, AS2.343;Saccharomyces logos van laer et Denamur ex Jorgensen, AS2.156, AS2.327,AS2.335; Saccharomyces mellis Lodder et Kreger Van Rij, AS2.195;Saccharomyces microellipsoides Osterwalder, AS2.699; Saccharomycesoviformis Osterwalder, AS2.100; Saccharomyces rosei (Guilliermond)Lodder et kreger van Rij, AS2.287; Saccharomyces rouxii Boutroux,AS2.178, AS2.180, AS2.370, AS2.371; Saccharomyces sake Yabe, ACCC2045;Candida arborea, AS2.566; Candida Krusei (Castellani) Berkhout,AS2.1045; Candida lambica(Linciner et Genoud) van.Uden et Buckley,AS2.1182; Candida lipolytica (Harrison) Diddens et Lodder, AS2.1207,AS2.1216, AS2.1220, AS2.1379, AS2.1398, AS2.1399, AS2.1400; Candidaparapsilosis (Ashford) Langeron et Talice, AS2.590; Candida parapsilosis(Ashford) et Talice Var. intermedia Van Rij et Verona, AS2.491; Candidapulcherriman (Lindner) Windisch, AS2.492; Candida rugousa (Anderson)Diddens et Loddeer, AS2.511, AS2.1367, AS2.1369, AS2.1372, AS2.1373,AS2.1377, AS2.1378, AS2.1384; Candida tropicalis (Castellani) Berkout,ACCC2004, ACCC2005, ACCC2006, AS2.164, AS2.402, AS2.564, AS2.565,AS2.567, AS2.568, AS2.617, AS2.1387; Candida utilis Henneberg Lodder etKreger Van Rij, AS2.120, AS2.281, AS2.1180; Crebrothecium ashbyii(Guillermond) Routein, AS2.481, AS2.482, AS2.1197; Geotrichum candidumLink, ACCC2016, AS2.361, AS2.498, AS2.616, AS2.1035, AS2.1062, AS2.1080,AS2.1132, AS2.1175, AS2.1183; Hansenula anomala (Hansen) H et P sydow,ACCC2018, AS2.294, AS2.295, AS2.296, AS2.297, AS2.298, AS2.299, AS2.300,AS2.302, AS2.338, AS2.339, AS2.340, AS2.341, AS2.470, AS2.592, AS2.641,AS2.642, AS2.635, AS2.782, AS2.794; Hansenula arabitolgens Fang,AS2.887; Hansenula jadinii Wickerham, ACCC2019; Hansenula saturnus(Klocker) H et P sydow, ACCC2020; Hansenula schneggi (Weber) Dekker,AS2.304; Hansenula subpelliculosa Bedford, AS2.738, AS2.740, AS2.760,AS2.761, AS2.770, AS2.783, AS2.790, AS2.798, AS2.866; Kloeckeraapiculata (Reess emend. Klocker) Janke, ACCC2021, ACCC2022, ACCC2023,AS2.197, AS2.496, AS2.711, AS2.714; Lipomyces starkeyi Lodder et vanRij, ACCC2024, AS2.1390; Pichia farinosa (Lindner) Hansen, ACCC2025,ACCC2026, AS2.86, AS2.87, AS2.705, AS2.803; Pichia membranaefaciensHansen, ACCC2027, AS2.89, AS2.661, AS2.1039; Rhodosporidium toruloidesBanno, ACCC2028; Rhodotorula glutinis (Fresenius) Harrison, ACCC2029,AS2.280, ACCC2030, AS2.102, AS2.107, AS2.278, AS2.499, AS2.694, AS2.703,AS2.704, AS2.1146; Rhodotorula minuta (Saito) Harrison, AS2.277;Rhodotorula rubar (Demme) Lodder, ACCC2031, AS2.21, AS2.22, AS2.103,AS2.105, AS2.108, AS2.140, AS2.166, AS2.167, AS2.272, AS2.279, AS2.282;Saccharomyces carlsbergensis Hansen, ACCC2032, ACCC2033, AS2.113,AS2.116, AS2.118, AS2.121, AS2.132, AS2.162, AS2.189, AS2.200, AS2.216,AS2.265, AS2.377, AS2.417, AS2.420, AS2.440, AS2.441, AS2.443, AS2.444,AS2.459, AS2.595, AS2.605, AS2.638, AS2.742, AS2.745, AS2.748, AS2.1042;Saccharomyces uvarum Beijer, IFFI 1023, IFFI 1032, IFFI 1036, IFFI 1044,IFFI 1072, IFFI 1205, IFFI 1207; Saccharomyces willianus Saccardo,AS2.5, AS2.7, AS2.119, AS2.152, AS2.293. AS2.381, AS2.392, AS2.434,AS2.614. AS2.1189: Saccharomyces sp., AS2.311 ; Saccharomyces ludwigiiHansen, ACCC2044, AS2.243, AS2.508; Saccharomyces sinenses Yue AS2.1395;Schizosaccharomyces octosporus Beijerinck, ACCC 2046, AS2.1148;Schizosaccharomyces pombe Linder, ACCC2047, ACCC2048, AS2.248, AS2.249,AS2.255, AS2.257, AS2.259, AS2.260, AS2.274, AS2.994, AS2.1043,AS2.1149, AS2.1178, IFFI 1056; Sporobolomyces roseus Kluyver et vanNiel, ACCC 2049, ACCC 2050, AS2.619, AS2.962, AS2.1036, ACCC2051,AS2.261, AS2.262; Torulopsis candida (Saito) Lodder, ACCC2052, AS2.270;Torulopsis famta (Harrison) Lodder et van Rij, ACCC2053, AS2.685;Torulopsis globosa (Olson et Hammer) Lodder et van Rij, ACCC2054,AS2.202; Torulopsis inconspicua Lodder et van Rij, AS2.75; Trichosporonbehrendii Lodder et Kreger van Rij, ACCC2055, AS2.1193; Trichosporoncapitatum Diddens et Lodder, ACCC2056, AS2.1385; Trichosporoncutaneum(de Beurm et al.)Ota, ACCC2057, AS2.25, AS2.570, AS2.571,AS2.1374; Wickerhamia fluoresens (Soneda) Soneda, ACCC2058, AS2.1388.

[0051] Certain yeast species that can be activated or induced accordingto the present invention and are included in the present invention areknown to be pathogenic to human and/or other living organisms, forexample, Ashbya gossypii; Blastomyces dermatitidis; Candida albicans;Candida parakrusei; Candida tropicalis; Citeromyces matritensis;Crebrothecium ashbyii; Cryptococcus laurentii; Cryptococcua neoformans;Debaryomyces hansenii; Debaryomyces kloeckeri; Debaryomyces sp.;Endomycopsis fibuligera. Under certain circumstances, it may be lesspreferable to use such pathogenic yeasts in the biological fertilizer ofthe invention, for example, if such use in an open field may endangerthe health of human and/or other living organisms.

[0052] Yeasts of the Saccharomyces genus are generally preferred. Amongstrains of Saccharomyces cerevisiae, Saccharomyces cerevisiae Hansen isa preferred strain. The most preferred strains of yeast areSaccharomyces cerevisiae strains having accession numbers AS2.504,AS2.558, AS2.413, AS2.397, AS2.69, AS2.109, AS2.607, AS2.516, AS2.561,AS2.422, AS2.393, AS2.631, AS2.982, AS2.560, AS2.467, AS2.415, AS2.375,AS2.628, AS2.1190, AS2.562, AS2.463, AS2.409, AS2.379, AS2.666, AS2.631,AS2.182, AS2.431, AS2.606, AS2.53, AS2.611, AS2.414, AS2.576, AS2.483,IFFI 1211, IFFI 1293, IFFI 1308, IFFI 1210, IFFI 1213, IFFI 1307, IFFI1206, IFFI 1052, IFFI 1301, IFFI 1291, IFFI 1202, IFFI 1021, IFFI 1059,IFFI 1052, IFFI 1441, IFFI 1008, IFFI 1220, IFFI 1302, and IFFI 1023 asdeposited at the China General Microbiological Culture Collection Center(CGMCC).

[0053] Generally, yeast strains useful for the invention can be obtainedfrom private or public laboratory cultures, or publically accessibleculture deposits,. such as the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209 and the China GeneralMicrobiological Culture Collection Center (CGMCC), China Committee forCulture Collection of Microorganisms, Institute of Microbiology, ChineseAcademy of Sciences, Haidian, P.O. Box 2714, Beijing, 100080, China.

[0054] The following yeast strains are preferred for making theP-balancing yeasts of the invention: AS2.558, AS2.118, AS2.103, AS2.132,AS2.121, AS2.189, AS2.216, AS2.265, AS2.417, AS2.420, AS2.200, AS2.162,AS2.440, AS2.277, AS2.441, AS2.443, AS2.444, AS2.605, AS2.595, AS2.638,AS2.742, AS2.748, AS2.14, AS2.16, AS2.56, AS2.69, AS2.70, AS2.109,AS2.112, AS2.375, AS2560, AS2.561, AS2.562, AS2.559, AS2.501, AS2.502,AS2.503, AS2.504, IFFI1001, IFFI1002, IFFI1005, IFFI1006, IFFI1008,IFFI1009, IFFI1010, IFFI1012, IFFI1021, IFFI1027, IFFI1037, IFFI1042,IFFI1060, IFFI1063, IFFI1202, IFFI1203, IFFI1206, IFFI1209, IFFI1210,IFFI1211, IFFI1212, IFFI1213, IFFI1215, IFFI1220, IFFI1221, IFFI1224,IFFI1247, IFFI1248, IFFI1251, IFFI1270, IFFI1277, IFFI1287, IFFI1289,IFFI1290, IFFI1291, IFFI1292, IFFI1293, IFFI1297, IFFI1300, IIFFI1301,IFFI1207, IFFI1307, IFFI1308, IFFI1309, IFFI1310, IFFI1311, IFFI1331,IFFI1335, IIFFI1336, IFFI1337, IFFI1338, IFFI1340, IFFI1339, IFFI1345,IFFI1396, IFFI1399, IFFI1411, IFFI1413, IFFI1023, IFFI1032, IFFI1036,IFFI1044, and IFFI1207.

[0055] Although it is preferred, the preparation of the yeast cellcomponents of the invention is not limited to starting with a purestrain of yeast. Each yeast cell component may be produced by culturinga mixture of yeast cells of different species or strains. Theconstituents of a yeast cell component can be determined by standardyeast identification techniques well known in the art.

[0056] Some yeasts may perform one of the desired functions moreefficiently than others. The table below lists the species and accessionnumbers of various yeast strains and the preferred functions for whichthe respective strains are stimulated by the methods of the invention.

[0057] The ability and efficiency of any species or strain of yeast toperform any one of the ten desired functions before or after culturingunder the conditions of the invention can readily be tested by methodsknown in the art. For example, the amount of nitrogen fixed can bedetermined by a modified acetylene reduction method as described in U.S.Pat. No. 5,578,486 which is incorporated herein by reference in itsentirety. The modified acetylene reduction method determines the amountof nitrogen fixed by measuring the decrease in molecular nitrogen in avolume of air. The amount of nitrogen fixed can also be determined bymeasurement of the ammonia and nitrates produced by the yeast cells(see, for example. Grewling et al., 1965, Cornell Agr Exp Sta Bull960:22-25). The amount of phosphorus available to plants as a result ofconversion from insoluble or biologically-unavailable phosphoruscompounds can be determined by the molybdenum blue method (see, forexample, Murphy et al., 1962, Analytica Chimica Acta 27:31-36) or the UVabsorption method; whereas the amount of available potassium convertedfrom insoluble or biologically-unavailable potassium compounds can bedetermined, for example, by flame atomic absorption spectroscopy (see,for example, Puchyr, et al., 1986, J. Assoc. Off. Anal. Chem.69:868-870). The ability of the yeasts to supply biologically availableN, P, and K after the biological fertilizer composition has been addedto soil can be tested by many techniques known in the art. For example,plant-available ammonia, nitrates, P, and K produced by the yeast cellsin soil can be extracted and quantitatively analyzed by the Morgan soiltest system (see, for example, Lunt et al., 1950, Conn Agr Exp Sta Bull541).

[0058] Methods well known in the art can be used for detecting andanalyzing various organic molecules in sludge, including HPLC.Similarly, methods well known in the art can be used for detecting andcounting the number of viable microorganisms and the total number ofmicroorganisms in a sample.

[0059] Without being bound by any theory or mechanism, the inventorbelieves that the culture conditions activate and/or enhance theexpression of a gene or a set of genes in a yeast cell such that thecell becomes active or more efficient in performing certain metabolicactivities which lead to the respective desired results.

[0060] The term “sludge” as used herein broadly encompasses any solidmatter that has settled out of suspension in the course of sewagestorage and/or treatment, for example, residues in a waste lagoon, in anurban sewage treatment plant. The term also include semi-solid matters,and mixtures of effluent and sediments. The term thus encompasses sludgehaving a wide range of viscosity, density, and water content, as well assludge which has been partially processed or stabilized.

[0061] Optionally, an inorganic substrate component can be included inthe biological fertilizer compositions of the invention. The inorganicsubstrate component can include but not limited to phosphate rock orrock phosphate, apatite, phosphorite, sylvinite, halite, carnalitite,and potassium mica.

[0062] Due to the variation of constituents in sludge, it may bedesirable to subject a sample of a batch of sludge to analysis todetermine the amount of plant nutrient present. Methods of soil analysiswell known in the art can be used to measure the amount of N, P, K,calcium, magnesium, zinc, iron, manganese, copper, sodium and sulfur inthe sludge.

[0063] In various embodiments, the biological fertilizer compositions ofthe present invention each comprises at least seven yeast cellcomponents capable of performing six basic functions plus at least oneof the supplementary functions. In a most preferred embodiment, thebiological fertilizer compositions comprise nine yeast cell components,in which case the six basic functions and all three supplementaryfunctions are provided. It will be understood that alternativeformulations are also contemplated.

[0064] In one particular embodiment of the invention, when a batch ofsludge that is relatively rich in biologically-available phosphorus isused, the biological fertilizer composition can be formulated tocomprise yeast cells that can maintain a balance of phosphorus compoundsinstead of yeast cells that decompose phosphorus-containing minerals orcompounds. Moreover, if desired, the biological fertilizer compositionmay comprise lesser quantities of one or more of the above-describedyeast cell components that supply one of the six basic functions. Forexample, if the biological fertilizer composition is to be used in soilthat is rich in potassium, the biological fertilizer composition can beformulated to comprise lesser amount of the yeast cells that candecompose potassium-containing minerals or compounds.

[0065] In another embodiment of the invention, where the yeast cells ofthe various yeast cell components are present in a mixture, the yeastcells can be cultured under certain conditions such that the yeast cellswith different functions can supply each other with and/or rely on eachother for nutrients and growth factors. As a result, a symbiosis-likerelationship is established among the various yeast cell components inthe fertilizer compositions of the invention. This culturing process isoptional but can improve the stability and efficiency of thecompositions such that the resulting fertilizer is made more suitablefor long term use in natural soil environments. The culturing conditionsfor this optional process are described in Section 5.11.

[0066] In yet another embodiment of the invention, the yeast cells mayalso be cultured under certain conditions so as to adapt the yeast cellsto a particular type of soil. This culturing process is optional, andcan be applied to each yeast cell component separately or to a mixtureof yeast cell components. The result is better growth and survival ofthe yeast cells in a particular soil environment. The culturingconditions for this optional process are described in Section 5.12.

[0067] As used herein, the biological fertilizer composition supports orenhances plant growth, if in the presence of the biological fertilizerin the soil, or applied to the roots, stems, leaves or other parts ofthe plant, the plant or a part of the plant gains viability, size,weight, rate of germination, rate of growth, or rate of maturation.Thus, the biological fertilizer compositions have utility in any kind ofagricultural, horticultural, and forestry practices. The biologicalfertilizer compositions can be used for large scale commercial farming,in open fields or in greenhouse, or even ill interiors for decorativeplants. Preferably, the biological fertilizer is used to enhance thegrowth of crop plants,.such as but not limited to cereal crops,vegetable crops, fruit crops, flower crops, and grass crops. Forexample, the biological fertilizer compositions may be used with wheat,barley, corn, soybean, rice, oat, potato, apple, orange, tomato, melon,cherry, lemon, lettuce, carrot, sugar cane, tobacco, cotton, etc.

[0068] The biological fertilizer compositions of the invention may beapplied in the same manner as conventional fertilizers. As known tothose skilled in the relevant art, many methods and appliances may beused. In one embodiment, culture broths of the yeast strains of thepresent invention are applied directly to soil or plants. In anotherembodiment, dried powders of the yeast strains of the present inventionare applied to soil or plants. In yet another embodiment, mixtures ofthe yeast cell components and organic substrate components of thepresent invention are applied to soil or plants. The biologicalfertilizer compositions may be applied to soil, by spreaders, sprayers,and other mechanized means which may be automated. The biologicalfertilizer compositions may be applied directly to plants, for example,by soaking seeds and/or roots, or spraying onto leaves. Such applicationmay be made periodically, such as once per year, or per growing season,or more frequently as desired. The biological fertilizer compositions ofthe invention can also be used in conjunction or in rotation with othertypes of fertilizers.

[0069] In one preferred embodiment, the biological fertilizercomposition of the invention, i.e., yeasts of the invention mixed withsludge in granular form, is used as a basal fertilizer which is appliedinto the soil at the depth of the major root system of the crop. Priorto application, the ground should be loosened and clear of weeds. Thebiological fertilizer composition can be spread evenly onto the ground,added to holes or long furrows in the ground. For existing fruit trees,a circular furrow of about 5 to 30 cm deep is dug into which thebiological fertilizer composition of the invention is added. The ground,holes, or furrows containing the biological fertilizer composition canthen be covered with soil and watered throughly. After 3 to 7 days, thearea is ready for planting or sowing. For rice, the ground is floodedwith water for 3 to 7 days before planting the seedlings. If used insandy soil with a shallow root system, a depth of 5 to 15 cm is used;with a deep root system, 5 to 25 cm is recommended. In clay soil with ashallow root system, a depth of 2 to 10 cm is used; with a deep rootsystem, 2 to 15 cm is recommended. The desired effect is that thebiological fertilizer composition is contact with or in very closeproximity to the roots of the plants. Preferably, after application ofthe fertilizer and/or planting, the soil is not disturbed. Generally,the operation temperature of the fertilizer is between 5° C. to 45° C.,optimally between 16° C. to 30° C.; the preferred pH range is between5.5 to 8.5, and optimally between 6.5 to 7.5. Recommended Dosage CropAmount of Biological Fertilizer Vegetables (short-growing) 600-900 kg/haVegetable (long-growing) 900-1200 kg/ha Ground vegetable 900-1350 kg/haSolanaceous fruit 900-1350 kg/ha Root & Tuber vegetable 750-900 kg/haBulb vegetable 900-1200 kg/ha Legume 600-1050 kg/ha Fruit Trees 2-5kg/tree Paddy Rice 600-900 kg/ha Wheat & Corn 750-1200 kg/ha Cotton &Peanut 600-1200 kg/ha

[0070] Described respectively in Sections 5.1-5.10 are the yeast cellcomponents used for nitrogen fixation, phosphorus compounddecomposition, potassium compound decomposition, complex carbon compounddecomposition, growth factors production, ATP production, pathogensuppression, degradation of undesirable chemicals, and reduction ofodor. Methods for preparing each yeast cell components are described.Section 5.11 describes the methods for establishing a symbiosis-likerelationship among yeast strains in a fertilizer composition of theinvention. Section 5.12 describes methods for adapting yeast cells ofthe invention to a particular type of soil. Section 5.13 describes themanufacture of the biological fertilizer compositions of the invention.Methods for the preparation of organic substrates and for themanufacture of the biological fertilizer, including mixing, drying,cooling, and packing, are also described. In various embodiments of theinvention, standard techniques for handling, transferring, and storingyeasts are used. Although it is not necessary, sterile conditions orclean environments are desirable when carrying out the processes of theinvention.

[0071] 5.1. Nitrogen-fixing Yeast Cell Component

[0072] Nitrogen fixation is a process whereby atmospheric nitrogen isconverted into ammonia and nitrates. Close to 800 species of naturallyoccurring microorganisms, mostly bacteria and cyanobacteria, from morethan 70 genera have been found to be able to fix nitrogen. Some of thenitrogen-fixing microorganisms, such as Rhizoboum, form symbioticassociation with plants, especially in the root of legumes. Others, suchas Azotobacter, are free-living and capable of fixing nitrogen in soil.

[0073] In the present invention, the ability of a yeast to fix nitrogenis activated or enhanced, and the resulting nitrogen-fixing yeast cellscan be used as a component of the biological fertilizer compositions ofthe invention.

[0074] According to the invention, yeast cells that have an enhancedability to fix nitrogen are prepared by culturing the cells in thepresence of an electromagnetic field in an appropriate culture medium.The frequency of the electromagnetic field for activating or enhancingnitrogen fixition in yeasts can generally be found within the range of800 MHz-1000 MHz. After the yeast cells have been cultured for asufficient period of time, the cells can be tested for their ability tofix nitrogen by methods well known in the art.

[0075] The method of the invention for making the nitrogen-fixing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0076] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, calcium, phosphate, sulfate, carbonate, and like ions.Non-limiting examples of nutrient inorganic salts are CaCO₃, KH₂PO₄,MgSO₄, NaCl, and CaSO₄. TABLE 1 Composition for a culture medium fornitrogen-fixing yeast Medium Composition Quantity KH₂PO₄ 0.2 g K₂HPO₄0.2 g MgSO₄.7H₂O 0.25 g CaCO₃.5H₂O 3.5 g CaSO₄.2H₂O 0.5 g NaCl 0.25 gYeast extract paste 0.3 g Sucrose 12.0 g Distilled water or autoclavedwater 1000 ml

[0077] It should be noted that the composition of the media provided inTable 1 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

[0078] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0079] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of about 800 to about 1000MHz, preferably in the range of 840.000 to 916.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 840, 845, 850, 855, 860, 865, 870, 875, 880, 885,890, 895, 900, 905, 910, 915, or 920 MHz. The field strength of the EMfield(s) is in the range of 10 to 200 mV/cm. If a series of EM fieldsare applied, the EM fields can each have a different frequency withinthe stated range, or a different field strength within the stated range,or different frequency and field strength within the stated ranges. In apreferred embodiment, the EM field(s) at the beginning of a series havea lower EM field strength than later EM field(s), such that the yeastcell culture are exposed to EM fields of progressively increasing fieldstrength. Although any practical number of EM fields can be used withina series, it is preferred that the yeast culture be exposed to a totalof 2, 3, 4, 5, 6, 7, or 8 different EM fields in a series.

[0080] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e.g., one or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 140-280hours.

[0081] For example, using an exemplary apparatus as depicted in FIG. 1,an initial EM field in the range of 10-20 mV/cm, usually at about 12.5mV/cm is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the EM field strength is increased to a higher levelin the range of 50-200 mV/cm, usually to about 125 mV/cm. The process ofthe invention is carried out at temperatures ranging from about 23° to30° C.; however, it is preferable to conduct the process at 25° to 28°C. The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

[0082] At the end of the culturing process, the nitrogen-fixing yeastcells may be recovered from the culture by various methods known in theart, and stored at a temperature below about 0° C. to 4° C. Thenitrogen-fixing yeast cells may also be dried and stored in powder form.

[0083] Any methods known in the art can be used to test the activatedyeast cells for their ability to fix nitrogen. For example, a modifiedacetylene reduction method for measuring nitrogen fixed bymicroorganisms is used to evaluate the nitrogen-fixing capability of theprepared yeast. The modified acetylene reduction method is described inU.S. Pat. No. 5,578,486 which is incorporated herein by reference in itsentirety. An alternative method based on 15-N can also be used.

[0084] The ability of the yeasts of the invention in fixing nitrogen canbe demonstrated by the following two methods:

[0085] One ml of activated yeast strain AS2.628 (2-5×10⁷) was culturedin 1000 ml of Ashby medium at 28° C. in the presence of a series of 8 EMfields in the order stated: 855 MHz at 14 mV/cm for 5 hours; 865 MHz at14 mV/cm for 5 hours; 875 MHz at 14 mV/cm for 5 hours; 885 MHz at 14mV/cm for 5 hours; 855 MHz at 120 mV/cm for 30 hours; 865 MHz at 120mV/cm for 30 hours; 875 MHz at 120 mV/cm for 30 hours; 885 MHz at 120mV/cm for 30 hours. In a separate container, as control, 1 ml ofnon-activated yeast was cultured under the same conditions without theEM fields. After culturing, the 1000 ml of yeast cells are mixed with3000 g sterilized coal dust powder, and then dried at less than 70° C.until the moisture content is less than 5%. The end product in powderform (0.1 g) was sealed with 10 ml of Ashby medium in a 100 ml cultureflask (5 flasks for each were used in the experiment). 10 ml of air wasremoved from the flasks by a syringe and replaced with 10 ml ofacetylene (>99% purity). The culture flasks were incubated at 28 ° C.for 24-120 hours and the amount of acetylene reduced was measured by gaschromatography. There was no significant reduction of acetylene in thecontrol containing non-activated yeasts.

[0086] Alternatively, the isotopic nitrogen dilution method can be used.The end product in powder form (0.1 g of non-activated and 0.1 g ofactivated yeasts) were cultured separately for 96 hours at 28° C. Theamount of nitrogen fixed by each was determined and compared. The amountof nitrogen fixed by activated yeasts was greater than 3.5 mg/g of thedried powder. The control containing non-activated yeasts did not showany significant fixation of nitrogen.

5.2. Phosphorus-decomposing Yeast Cell Component

[0087] The phosphorus compound-decomposing (P-decomposing) yeast of theinvention converts insoluble or biologically-unavailablephosphorus-containing substances, such as phosphate rock, into solublephosphorous compounds so that they become available to plants.

[0088] In the present invention, the ability of yeasts to decomposeinsoluble phosphorus-containing substances is activated or enhanced, andthe resulting P-decomposing yeast cells can be used as a component ofthe biological fertilizer compositions of the invention.

[0089] In various embodiments, P-decomposing yeast cells are employed inthe compositions of the invention when the level of soluble orbiologically-available phosphorous is low in the organic substrate (forexample, cattle manure, swine manure, sludge and garbage).

[0090] According to the invention, yeast cells that are capable ofP-decomposing are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate culture medium. The frequency ofthe electromagnetic field for activating or enhancing P-decomposition inmicrobes can generally be found in the range of 300 MRz to 500 MHz.After the cells have been cultured for a sufficient period of time, thecells can be tested for their ability to decompose phosphorus-containingsubstances by methods well known in the

[0091] The method of the invention for making the P-decomposing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0092] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, calcium, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are CaCO₃, MgSO₄, NaCl, and CaSO₄.Non-biologically available forms of phosphorus-containing substances ina suitable form are also included in the media as dried organicsubstrate. Non-limiting examples of dried organic substrate includemanure, sludge and garbage of ≧150 mesh. Other insolublephosphorus-containing substances can also be used either separately orin combination. TABLE 2 Composition for a culture medium forP-decomposing yeast Medium Composition Quantity Sucrose 15 g NaCl 1.2 gMgSO₄.7H₂O 0.2 g CaCO₃.5H₂O 3.0 g CaSO₄.2H₂O 0.3 g KNO₃ 0.3 g Yeastextract paste 0.5 g Dried sludge 1.2 g to 2.4 g: Powder of > 150 meshAutoclaved water 1000 ml

[0093] It should be noted that the composition of the media provided inTable 2 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations such as the scale of culture andlocal supply of media components.

[0094] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0095] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of about 300 to about 500MHz, preferably in the range of 340.000 to 435.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 340, 345, 350, 355, 360, 365, 370, 375, 375, 380,385, 390, 395, 400, 405, 410, 415, 420, 425, 430 or 435 MHz. The fieldstrength of the EM field(s) is in the range of 10 to 200 mV/cm. If aseries of EM fields are applied, the EM fields can each have a differentfrequency within the stated range, or a different field strength withinthe stated range, or different frequency and field strength within thestated ranges. In a preferred embodiment, the EM field(s) at thebeginning of a series have a lower EM field strength than later EMfield(s), such that the yeast cell culture are exposed to EM fields ofprogressively increasing field strength. Although any practical numberof EM fields can be used within a series, it is preferred that the yeastculture be exposed to a total of 2, 3, 4, 5, 6, 7, or 8 different EMfields in a series.

[0096] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e.g., one or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 140-280hours.

[0097] For example, using an exemplary apparatus as depicted in FIG. 1,an initial field strength in the range of 10-20 mV/cm, usually at about12.5 mV/cm is used. After this first period of culture, the yeast cellsare further incubated under substantially the same conditions foranother period, except that the EM field strength is increased to ahigher level in the range of 50-200 mV/cm, Usually to about 125 mV. Theprocess of the invention is carried out at temperatures ranging fromabout 23° to 30° C.; however, it is preferable to conduct the process at25° to 28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

[0098] At the end of the culturing process, the P-decomposing yeastcells may be recovered from the culture by various methods known in theart, and stored at a temperature below about 0° C. to 4° C. TheP-decomposing yeast cells may also be dried and stored in powder form.

[0099] The amount of biologically available phosphorus, such as H₃PO₄,H₂PO₄ ⁻, and HPO₄ ²⁻, in the culture can be determined by any methodsknown in the art, including but not limited to UV absorptionspectroscopy. The increase can be calculated by the difference betweenthe total amount of biologically available phosphorus in a culture withactivated yeasts and the amount of biologically available phosphorus inthe same medium with non-activated yeast. For example, 1 ml ofSaccharomyces cerevisiae strain AS2.399 (2 to 5×10⁷ yeasts/ml) isinoculated into 1000 ml of a medium according to Table 2. The culture isincubated at a temperature of 28° C. in the presence of a series of 8 EMfields in the order stated: 360 MHz at 14 mV/cm for 5 hours; 365 MHz at14 mV/cm for 5 hours; 370 MHz at 14 mV/cm for 5 hours; 380 MHz at 14mV/cm for 5 hours; 360 MHz at 130 mV/cm for 30 hours; 365 MHz at 130mV/cm for 30 hours; 370 MHz at 130 mV/cm for 30 hours; 375 MHz at 130mV/cm for 30 hours. The increase in the amount of biologically availablephosphorus was determined to be greater than 330 mg/ml of yeast culture.

[0100] 5.3. Phosphorus-Balancing Yeast Cell Component

[0101] The phosphorus-balancing (P-balancing) yeasts of the inventionalso convert insoluble or biologically unavailable phosphorus-containingsubstances into soluble biologically available phosphorous compounds.However, the P-balancing yeast is preferably used when the level ofphosphorus in the local environment is high The conversion of insolubleor biologically inavailable phosphorus-containing substances intosoluble biologically available phosphorous is sensitive to the level ofphosphorus; at about 180 ppm or higher, the conversion is reduced whileat about 60 ppm or lower, the conversion is increased.

[0102] In the present invention, the P-balancing yeast cells arepreferably deployed in biologically fertilizer compositions that includean organic substrate that already contains a relatively significantlevel of soluble or biologically available phosphorous. For example,sludge contains a relatively high level of soluble phosphorus ascompared to other kinds of manure.

[0103] According to the invention, yeast cells that are capable ofP-balancing are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate. culture medium. The frequencyof the electromagnetic field for activating or enhancing P-balancingfunction in yeasts can generally be found in the range of 300 MHz to 500MHz. After the cells have been cultured for a sufficient period of time,the cells can be tested for their ability to decomposephosphorus-containing substances by methods well known in the art.

[0104] The method of the invention for making the P-balancing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0105] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, calcium, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are CaCO₃, MgSO₄, NaCl, and CaSO₄.Insoluble phosphorus-containing substances in a suitable form are alsoincluded in the media. Non-limiting examples include powder of driedsludge of >150 mesh. Other insoluble phosphorus-containing substancescan also be used either separately or in combination. TABLE 3Composition for a culture medium for P-balancing yeast MediumComposition Quantity Sucrose 15 g NaCl 1.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O3.0 g CaSO₄.2H₂O 0.3 g KNO₃ 0.3 g Yeast extract paste 0.5 g Dried sludge1.2 g; Powder of > 150 mesh Autoclaved water 1000 ml

[0106] It should be noted that the composition of the media provided inTable 3 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

[0107] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0108] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of about 300 to about 500MHz, or preferably in the range of 380.000 to 485.000 MHz. For exampleand without being limited by such examples, each EM field can have afrequency at about 380, 385, 390, 395, 400, 402, 405, 410, 415, 420,422, 425, 430, 432, 435, 440, 445, 450, 455, 460, 465, 470, 480 or 485MHz. The field strength of the EM field(s) is in the range of 90 to 300mV/cm. If a series of EM fields are applied, the EM fields can each havea different frequency within the stated range, or a different fieldstrength within the stated range, or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7 or 8 different EM fields in a series.

[0109] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e.g., two or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 230-480hours.

[0110] For example, using an exemplary apparatus as depicted in FIG. 1,an initial field strength in the range of 50-150 mV/cm, usually at about100 mV is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the EM field strength is increased to a higher levelin the range of 200-300 mV/cm, usually to about 250 mV/cm. The processof the invention is carried out at temperatures ranging from about 23°to 30° C.; however, it is preferable to conduct the process at 25° to28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

[0111] At the end of the culturing process, the P-balancing yeast cellsmay be recovered from the culture by various methods known in the art,and stored at a temperature below about 0° C. to 4° C. The P-balancingyeast cells may also be dried and stored in powder form.

[0112] The amount of biologically available phosphorus, such as H₃PO₄,H₂PO₄ ⁻, and HPO₄ ²⁻, in the culture can be determined by any methodsknown in the art, including but not limited to UV absorptionspectroscopy. The increase can be calculated by the difference betweenthe total amount of biologically available phosphorus in a culture withactivated yeasts and the amount of phosphorus in the same medium withnon-activated yeast. For example, 1 ml of Saccharomyces cerevisiaestrain AS2.628 (2 to 5×10⁷ yeasts/ml) is inoculated into 1000 ml of amedium containing 200 mg/l of H₃PO₄, H₂PO₄ and HPO₄ ²⁻. The culture isincubated at a temperature of 28° C. in the presence of a series of 8 EMfields in the order stated: 385 MHz at 99 mV/cm for 12 hours; 415 MHz at99 mV/cm for 12 hours; 440 MHz at 99 mV/cm for 12 hours; 460 MHz at 99mV/cm for 12 hours; 385 MHz at 250 mV/cm for 48 hours; 415 MHz at 250mV/cm for 48 hours; 440 MHz at 250 mV/cm for 24 hours; 460 MHz at 250mV/cm for 24 hours. The increase in the amount of biologically availablephosphorus was determined to be greater than 24%. The control did notshow any significant change in the amount of biologically as availablephosphorus.

[0113] 5.4. Potassium-Decomposing Yeast Cell Component

[0114] The potassium compound-decomposing (K-decomposing) yeasts of theinvention converts insoluble potassium-containing substances, such aspotassium mica, into soluble potassium so that they become available toplants.

[0115] In the present invention the ability of a plurality of yeastcells to decompose insoluble potassium-containing substances isactivated or enhanced, and the resulting K-decomposing yeast cells canbe used as a component of the biological fertilizer compositions of theinvention.

[0116] According to the present invention, yeast cells that are capableof K-decomposing are prepared by culturing the cells in the presence ofan electromagnetic field in an appropriate culture medium. The frequencyof the electromagnetic field for activating or enhancing K-decompositionin yeasts can generally be found in the range of 100 MHz -300 MHz. Afterthe yeast cells have been cultured for a sufficient period of time, thecells can be tested for their ability to decompose potassium-containingsubstances by methods well known in the art.

[0117] The method of the invention for making the K-decomposing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0118] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are (NH₄)₂HPO₄, CaCO₃, MgSO₄, NaCl,and CaSO₄. Insoluble potassium-containing substances in a suitable formare also included in the media. Non-limiting examples include powder ofpotassium mica of ≧200 mesh. Other insoluble potassium-containingsubstances can also be used either separately or combined. TABLE 4Composition for a culture medium for K-decomposing yeast MediumComposition Quantity Sucrose 15 g NaCl 1.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O3.0 g CaSO₄.2H₂O 0.3 g (NH₄)₂HPO₄ 0.3 g Yeast extract paste 0.5 gPotassium mica 1.0 g, Powder of > 200 mesh Dried sludge 1.2-3 g, Powderof > 150 mesh Autoclaved water 1000 ml

[0119] It should be noted that the composition of the media provided inTable 4 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

[0120] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0121] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of about 100 to about 300MHz, preferably in the range of 190.000 to 285.000 MHz. For example andwithout being limited by such examples, each EM field can have afrequency at about 190, 195, 200, 205, 210, 215, 220, 225, 230, 235,240, 245, 250, 255, 260, 265, 270, 275, 280, or 285 MHz. The fieldstrength of the EM field(s) is in the range of 10 to 200 mV/cm If aseries of EM fields are applied, the EM fields can each have a differentfrequency within the stated range, or a different field strength withinthe stated range, or different frequency and field strength within thestated ranges. In a preferred embodiment, the EM field(s) at thebeginning of a series have a lower EM field strength than later EMfield(s), such that the yeast cell culture are exposed to EM fields ofprogressively increasing field strength. Although any practical numberof EM fields can be used within a series, it is preferred that the yeastculture be exposed to a total of 2, 3, 4, 5, a, 7, or 8 different EMfields in a series.

[0122] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e.g., one or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 140-280hours.

[0123] For example, using an exemplary apparatus as depicted in FIG. 1,an initial field strength in the range of 10-20 mV/cm, usually at about125 mV/cm is used. After this first period of culture, the yeast cellsare further incubated under substantially the same conditions foranother period, except that the EM field strength is increased to ahigher level in the range of 50-200 mV/cm, usually to about 125 mV/cm.The process of the invention is carried out at temperatures ranging fromabout 23° to 30° C.; however, it is preferable to conduct the process at25° to 28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

[0124] At the end of the culturing process, the K-decomposing yeastcells may be recovered from the culture by various methods known in theart, and stored at a temperature below about 0-4° C. The K-decomposingyeast cells may also be dried and stored in powder form.

[0125] Any methods known in the art can be used to test the culturedyeast cells for their ability to decompose insolublepotassium-containing substances. For example, 1 ml of Saccharomycescerevisiae strain AS2.631 (2 to 5×10⁷ cells/ml) was inoculated into 1000ml of a medium according to Table 4. The culture was incubated at atemperature at 28° C. in the presence of a series of 8 EM fields in theorder stated: 210 MHz at 14 mV/cm for 5 hours; 235 MHz at 14 mV/cm for 5hours; 245 MHz at 14 mV/cm for 5 hours; 255 MHz at 14 mV/cm for 5 hours;210 MHz at 120 mV/cm for 30 hours; 235 MHz at 120 mV/cm for 30 hours;245 MHz at 120 mV/cm for 30 hours; 255 MHz at 120 mV/cm for 30 hours. Acontrol was set up which contained non-activated cells of the samestrain of yeasts. The amount of biologically available potassium K⁺inthe culture can be determined by any methods known in the art, includingbut not limited to flame spectroscopy and/or atomic absorptionspectrometry. The increase in potassium is calculated by the differencebetween the quantity of potassium in the medium of Table 4 afterculturing and the basal level of potassium in the medium prior toculturing. The increase in the amount of biologically availablepotassium was determined to be greater than 120 mg/ml of cultured yeastcells. There was no significant change in the amount of potassiumavailable in the control.

[0126] 5.5. Complex Carbon-decomposing Yeast Cell Component

[0127] The carbon-decomposing (C-decomposing) yeast of the inventionconverts complex, high molecular weight, carbon compounds and materials,in particular, complex carbohydrates, such as cellulose and lignin, intosimple carbohydrates, such as pentoses and hexoses. Such simplecarbohydrates are utilized by other yeast cells in the compositions tosupport their growth and activities.

[0128] In a preferred embodiment, yeast cells are used to make theC-decomposing yeast cell component of the invention. In the presentinventions the ability of yeast to decompose complex carbon compoundsefficiently is activated or enhanced, and the resulting C-decomposingyeast cells can be used as a component of the biological fertilizercomposition of the invention.

[0129] According to the present invention, yeast cells that are capableof C-decomposition are prepared by culturing the cells in the presenceof an electromagnetic field in an appropriate culture medium. Thefrequency of the electromagnetic field for C-decomposition in yeasts cangenerally be found in the range of 1000 MHz-1200 MHz. After the yeastcells have been cultured for a sufficient period of time, the cells canbe tested for their ability to decompose complex carbon compounds bymethods well known in the art.

[0130] The method of the invention for making the C-decomposing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. Complex carbon-containingsubstances such as cellulose, lignin, coal powder, etc., in a suitableform can be used as sources of carbon in the culture medium. The exactquantity of the carbon source or sources utilized in the medium dependsin part upon the other ingredients of the medium but, in general, theamount of simple carbohydrate usually varies between about 0.1% and 5%by weight of the medium and preferably between about 0.1% and 1%, andmost preferably about 0.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0131] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃,MgSO₄, NaCl, and CaSO₄. TABLE 5 Composition for a culture medium forC-decomposing yeasts Medium Composition Quantity Cellulose 3.0 g; Powderof > 100 mesh Dried sludge 5 g; Powder of > 150 mesh NaCl 0.6 gMgSO₄.7H₂O 0.3 g CaCO₃.5H₂O 1.5 g CaSO₄.2H₂O 0.4 g (NH₄)₂HPO₄ 0.3 gYeast extract paste 0.5 g K₂HPO₄ 0.5 g Autoclaved water 1000 ml

[0132] It should be noted that the composition of the media provided inTable 5 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

[0133] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0134] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of about 1000 to about 1200MHz, preferably in the range of 1050.000 to 1160.000 MHz. For exampleand without being limited by such examples, each EM field can have afrequency at about 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090,1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135, 1140, 1145. 1150,1155, or 1160 MHz. The field strength of the EM field(s) is in the rangeof 10 to 200 mV/cm. If a series of EM fields are applied, the EM fieldscan each have a different frequency within the stated range, or adifferent field strength within the stated range, or different frequencyand field strength within the slated ranges. In a preferred embodiment,the EM field(s) at the beginning of a series have a lower EM fieldstrength than later EM field(s), such that the yeast cell culture areexposed to EM fields, of progressively increasing field strength.Although any practical number of EM fields can be used within a series,it is preferred that the yeast culture be exposed to a total of 2, 3, 4,5, 6, 7, or 8 different EM fields in a series

[0135] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e.g., one or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 140-280hours.

[0136] For example, using an exemplary apparatus as depicted in FIG. 1,an initial field strength in the range of 10-20 mV, usually at about12.5 mV/cm is used. After this first period of culture, the yeast cellsare further incubated under substantially the same conditions foranother period, except that the EM field strength is increased to ahigher level in the range of 100-200 mV/cm, usually to about 125 mV/cm.The process of the invention is carried out at temperatures ranging fromabout 23° to 30° C.; however, it is preferable to conduct the process at25° to 28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

[0137] At the end of the culturing process, the C-decomposing yeastcells may be recovered from the culture by various methods known in theart, and stored at a temperature below about 0-4° C. The C-decomposingyeast cells may also be dried and stored in powder form.

[0138] Any methods known in the art can be used to test the culturedyeast cells for their ability to decompose complex-carbon containingsubstances. For example, a change in the chemical oxygen demand (COD) ofa sample can be used as an indication of the change in the concentrationof complex-carbon containing substances in the sample. For example, 1 mlof the Saccharomyces cerevisiae strain AS2 982 (2 to ×10⁷ yeasts/ml) isinoculated into 30 ml of a medium according to Table 5. The culture isincubated at a temperature in the range of 20-28 ° C. for in thepresence of a series of 8 EM fields in the order stated: 1050 MHz at 16mV/cm for 5 hours; 1070 MHz at 16 mV/cm for 5 hours; 1090 MHz at 16mV/cm for 5 hours; 1110 MHz at 16 mV/cm for 5 hours 1, 050 MHz at 125mV/cm for 30 hours;1070 MHz at 125 mV/cm for 30 hours; 1090 ,MHz at 125mV/cm for 30 hours; 1110 MHz at 125 mV/cm for 30 hours. Afteractivation, based on a change in COD, the amount of carbohydrates in theculture was estimated to be greater than 330 mg/ml of yeast culture.

[0139] Alternatively, the amount of simple carbohydrates in the culturecan then he determined by any methods known in the art. including butnot limited to biochemical reactions, chromatography and molecularfluorescence spectroscopy.

[0140] 5.6. Growth Factors Producing Yeast Cell Component

[0141] The growth factors producing (GF-producing) yeast of the presentinvention produces many vitamins and other nutrients, such as but notlimited to, vitamin B-1, riboflavin (vitamin B-2), vitamin B-12, niacin(B-3), pyridoxine (B-6), pantothenic acid (B-5), folic acid, biotin,para-aminobenzoic acid, choline, inositol, in such amounts that cansupport the growth of other yeast strains.

[0142] The ability of yeast to overproduce growth factors is activatedor enhanced by methods of this invention, and the resulting GF-producingyeast cells are included as a component of the biological fertilizercomposition of the invention.

[0143] According to the present invention, yeast cells that are capableof overproducing growth factors are prepared by culturing the yeastcells in the presence of an electromagnetic field in an appropriateculture medium. The frequency of the electromagnetic field foractivating or enhancing GF-production in yeasts can generally be foundin the range of 1300 MHz -1500 MHz. After the yeast cells have beencultured for a sufficient period of time, the cells can be tested fortheir ability to produce growth factors by methods well known in theart.

[0144] The method of the invention for making the GF-producing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0145] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are NH₄NO₃, K₂HPO₄, CaCO₃, MgSO₄,NaCl, and CaSO₄. TABLE 6 Composition for a culture medium forGF-producing yeasts Medium Composition Quantity Starch 8.0 g; Powderof > 120 mesh NaCl 0.3 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O 0.5 g CaSO₄.2H₂O0.2 g NH₄NO₃ 0.3 g K₂HPO₄ 0.8 g Autoclaved water 1000 ml

[0146] It should be noted that the composition of the media provided inTable 6 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

[0147] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0148] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of about 1300 to about 1500MHz, preferably in the range of 1340.000 to 1440.000 MHz. For exampleand without being limited by such examples, each EM field can have afrequency at about 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380,1385, 1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435. or1440 MHz. The field strength of the EM field(s) is in the range of 20 to200 mV/cm. If a series of EM fields are applied, the EM fields can eachhave a different frequency within the stated range, or a different fieldstrength within the stated range, or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7 or 8 different EM fields in a series.

[0149] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e.g., one or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 140-280hours.

[0150] For example, using an exemplary apparatus as depicted in FIG. 1,an initial field strength in the range of 20-40 mV/cm, usually at about25 mV/cm is used. After this first period of culture, the yeast cellsare further incubated under substantially the same conditions foranother period, except that the amplitude is increased to a higher levelin the range of 100-200 mV/cm, usually to about 125 mV. The process ofthe invention is carried out at temperatures ranging from about 23° to30° C.; however, it is preferable to conduct the process at 25° to 28°C. The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

[0151] At the end of the culturing process, the GF-producing yeast cellsmay be recovered from the culture by various methods known in the art,and stored at a temperature below about 0-4° C. The GF-producing yeastcells may also be dried and stored in powder form.

[0152] Any methods known in the art can be used to test the culturedyeast cells for their ability to overproduce growth factors, includingbut not limited to high performance liquid chromatography (HPLC). Forexample, 1 ml of activated or non-activated Saccharomyces cerevisiaestrain AS2.413 (2 to 5×10⁷ yeasts/ml) was inoculated into 1000 ml of amedium according to Table 6. The culture was incubated at a temperatureof 28 ° C. in the presence of a series of 8 EM fields in the orderstated: 1340 MHz at 28 mV/cm for 5 hours; 1350 MHz at 28 mV/cm for 5hours; 1380 MHz at 28 mV/cm for 5 hours; 1390 MHz at 28 mV/cm for 5hours; 1340 MHz at 135 mV/cm for 30 hours; 1350 MHz at 135 mV/cm for 30hours; 1380 MHz at 135 mV/cm for 30 hours; 1390 MHz at 135 mV/cm for 30hours. The amount of growth factors produced can be calculated by thedifference between the total amount of vitamin B1, B2 B6, B12 in aculture with activated or non-activated yeasts and the total amount ofthe same growth factors in the same medium without yeast. The increasein the amount of growth factors was determined to be greater than 350mg/ml of activated yeast culture.

[0153] 5.7. ATP-Producing Yeast Cell Component

[0154] The ATP-producing yeast of the present invention is capable ofoverproducing ATP in such amounts that can support the growth of othermicrobes in the biological fertilizer compositions.

[0155] In the present invention, the ability of yeast to overproduce ATPis activated or enhanced, and the resulting ATP-producing yeast cellscan be used as a component of the biological fertilizer compositions ofthe invention.

[0156] According to the present invention, yeast cells that are capableof enhanced ATP-production are prepared by culturing the cells in thepresence of an electric field in an appropriate culture medium. Thefrequency of the electromagnetic field for activating or enhancingATP-production in yeasts can generally be found in the range of 1600 MHz-1800 MHz. After sufficient time is given for the cells to grow, thecells can be tested for their enhanced ability to produce ATP by methodswell known in the art.

[0157] The method of the invention for making the ATP-producing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0158] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are (NH₄)₂HPO₄, K₂HPO₄ , CaCO₃,MgSO₄, NaCl, and CaSO₄. TABLE 7 Composition for a culture medium forATP-producing yeasts Medium Composition Quantity Starch 10.0 g, 120 >mesh NaCl 0.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O 0.8 g CaSO₄.2H₂O 0.2 gNH₄NO₃ 0.2 g K₂HPO₄ 0.5 g Autoclaved water 1000 ml

[0159] It should be noted that the composition of the media provided inTable 7 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

[0160] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0161] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of about 1600 to about 1800MHz, preferably in the range of 1630.000 to 1730.000 MHz. For exampleand without being limited by such examples, each EM field can have afrequency at about 1630, 1635, 1640, 1645, 1650, 1655, 1660, 1665, 1670,1675, 1680, 1685, 1690, 1695, 1700, 1705, 1710, 1715, 1720, 1725, or1730 MHz. The field strength of the EM field(s) is in the range of 20 to200 mV/cm. If a series of EM fields are applied, the EM fields can eachhave a different frequency within the stated range, or a different fieldstrength within the stated range, or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7, or 8 different EM fields in a series.

[0162] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod or time (e.g., one or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 160-300hours.

[0163] For example, using an exemplary apparatus as depicted in FIG. 1,an initial field strength in the range of 20-40 mV/cm, usually at about30 mV/cm is used. After this first period of culture, the yeast cellsare further incubated under substantially the same conditions foranother period, except that the amplitude is increased to a higher levelin the range of 100-200 mV/cm, usually to about 150 mV/cm. The processof the invention is carried out at temperatures ranging from about 23°to 30° C.; however, it is preferable to conduct the process at 25° to28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

[0164] At the end of the culturing process, the ATP-producing yeastcells may be recovered from the culture by various methods known in theart, and stored at a temperature below about 0-4° C. The ATP-producingyeast cells may also be dried and stored in powder form.

[0165] Any methods known in the art can be used to test the culturedyeast cells for their ability to overproduce ATP, including but notlimited to HPLC. For example, 1 ml of the activated yeast culture (2 to5×10⁷ yeasts/ml) was inoculated into 1000 ml of a medium according toTable 7. The culture was incubated at a temperature of 28° C. in thepresence of a series of 8 EM fields in the order stated: 1635 MHz at 29mV/cm for 10 hours; 1655 MHz at 29 mV/cm for 10 hours; 1675 MHz at 29mV/cm for 10 hours; 1695 MHz at 29 mV/cm for 10 hours; 1635 MHz at 150mV/cm for 30 hours; 1655 MHz at 150 mV/cm for 30 hours; 1675 MHz at 150mV/cm for 30 hours; 1695 MHz at 150 mV/cm for 30 hours. The amount ofATP produced can be calculated by the difference between the totalamount of ATP in a culture with yeasts and the amount of ATP in the samemedium without yeast. Using activated Saccharomyces cerevisiae strainAS2.536, the amount of ATP in the culture was determined to be 170 mg/mlof yeast culture.

[0166] 5.8 Pathogen-suppressing Yeast Cell Component

[0167] The present invention also provides yeast cells that are capableof suppressing the proliferation of pathogenic microorganisms that arepresent in the materials used in the organic substrate component of thebiological fertilizer Typically, due to an abundance of nutrientspresent in the organic substrate material for such pathogenicmicroorganisms, the numbers of pathogens increase rapidly over a periodof time. However, in the presence of the pathogen-suppressing yeasts ofthe invention, the numbers of pathogens in the organic substratematerial remains unchanged, or decreases over time. Without being boundby any theory or mechanism, the inventor believes that the presence ofthe pathogen-suppressing yeasts in the organic substrate materialcreates an environment that is unfavorable for the growth of pathogenicmicroorganisms.

[0168] According to the invention, the ability of yeasts toaffect/control the numbers of pathogens is activated or enhanced byculturing the yeasts in the presence of an electromagnetic field. Theresulting pathogen-suppressing yeast cells are used as a component inthe biological fertilizer compositions of the invention.

[0169] The frequency of the electromagnetic field for activating orenhancing the ability of yeasts to control the numbers of pathogenicmicroorganisms can generally be found in the range of 30 MHz to 50 MHz.After sufficient time is given for the yeast cells to grow, the cellscan be tested for their ability to affect/control the number ofpathogens by methods well known in the art.

[0170] The method of the invention for making pathogen-suppressing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%.and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0171] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃,MgSO₄, NaCl and CaSO,₄. TABLE 8 Composition for a culture medium forPathogen-Suppressing yeasts Medium Composition Quantity Soluble Starch8.0 g Sucrose 5 g NaCl 0.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O 0.5 gCaSO₄.2H₂O 0.2 g Peptone 1.5 g K₂HPO₄ 0.5 g Autoclaved water 400 mlSludge extract 600 ml

[0172] The sludge extract for the culture medium is prepared byincubating 500 g of sludge in about 600 ml of warm water (at 35° C. to40° C.) for 24 hours at 30-37° C., and filtering the fluid to removeparticulate matters. It should be noted that the composition of themedia provided in Table 8 is not intended to be limiting. Variousmodifications of the culture medium may be made by those skilled in theart, in view of practical and economic considerations, such as the scaleof culture and local supply of media components.

[0173] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0174] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of about 30.000 to about50.000 MHz. For example and without being limited by such examples eachEM field can have a frequency at about 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 MHz. The fieldstrength of the EM field(s) is in the range of 0.5 to 200 mV/cm,preferably 10 to 180 mV/cm. If a series of EM fields are applied, the EMfields can each have a different frequency within the stated range, or adifferent field strength within the stated range or different frequencyand field strength within the stated ranges. In a preferred embodiment,the EM field(s) at the beginning of a series have a lower EM fieldstrength than later EM field(s), such that the yeast cell culture areexposed to EM fields of progressively increasing field strength.Although any practical number of EM fields can be used within a series,it is preferred that the yeast culture be exposed to a total of 2, 3, 4,5, 6, 7, or 8 different EM fields in a series.

[0175] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e.g., one or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 144-272hours.

[0176] For example, using an exemplary apparatus as depicted in FIG. 1,an initial field strength in the range of 10-30 mV/cm, usually at about25 mV/cm is used. After this first period of culture, the yeast cellsare further incubated under substantially the same conditions foranother period, except that the amplitude is increased to a higher levelin the range of 100-200 mV/cm, usually to about 150 mV/cm. The processof the invention is carried out at temperatures ranging from about 23°to 30° C.; however, it is preferable to conduct the process at 25° to28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/M³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

[0177] At the end of the culturing process, the pathogen-suppressingyeast cells may be recovered from the culture by various methods knownin the art, and stored at about 0° C. to 4° C. The pathogen-suppressingyeast cells may also be dried and stored in powder form.

[0178] The ability of the pathogen-suppressing yeasts to control thenumbers of pathogens can be determined by any methods known in the artfor enumerating microorganisms, such as optical density, plating outdilutions on solid media for counting, or counting individual cellsunder a microscope. Stains may be applied to distinguish or identifydifferent strains or species of microorganisms present in a sample, orto determine their viability. When a range of pathogenic microorganismsare expected to be affected by the pathogen-suppressing yeasts, thenumbers of move than one representative species of pathogenicmicroorganisms can be monitored to assess the performance of thepathogen-suppressing yeasts.

[0179] For example, samples of organic substrate material containing aknown concentration of pathogenic microorganisms are cultured under thesame conditions for a same period of time in the presence of differentconcentrations of pathogen-suppressing yeasts, and as negative control,the same strain of yeasts that have not been treated according to theculturing methods of the invention. A sample without any added yeast mayalso be included to determine the growth of pathogens under normalcircumstances. The numbers of pathogens before and after the cultureperiod are determined and compared.

[0180] A one liter culture containing at least 10¹⁰ cells of apathogenic microorganism per ml is prepared. One ml of activated yeastcells (containing 2 to 5×10⁷ yeasts per ml) is added to the one literculture of pathogenic microorganism and incubated at 30° C. for 24hours. Controls are included which contained non-activated yeast cellsor no yeasts. The numbers of microorganisms in the respective culturesare then determined and compared. The following are several examples inwhich a particular species of pathogenic bacteria was studied.

[0181] Using cells of Saccharomyces cerevisiae strain IFFI1037 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 30 MHz at 26 mV/cm for 12 hours; 36 MHz at 26 mV/cm for 12hours; 43 MHz at 26 mV/cm for 12 hours; 47 MHz at 26 mV/cm for 12 hours;30 MHz at 150 mV/cm for 24 hours; 36 MHz at 150 mV/cm for 24 hours; 43MHz at 150 mV/cm for 24 hours; 47 MHz at 150 mV/cm for 24 hours. Thenumber of Staphylococcus aureus in a sample was reduced by more than2.7% relative to the control with no yeasts. There was no significantchange in the number of pathogens in the control containingnon-activated cells.

[0182] Using cells of Saccharomyces cerevisiae strain IFFI1021 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 30 MHz at 26 mV/cm for 12 hours; 36 MHz at 26 mV/cm for 12hours; 42 MHz at 26 mV/cm for 12 hours; 49 MHz at 26 mV/cm for 12 hours;30 MHz at 150 mV/cm for 24 hours; 36 MHz at 150 mV/cm for 24 hours, 42MHz at 150 mV/cm for 24 hours; 49 MHz at 150 mV/cm for 24 hours. Thenumber of Diplococcus pneumoniae in a sample was reduced by more than2.8% relative to the control with no yeasts. There was no significantchange in the number of pathogens in the control containingnon-activated cells.

[0183] Using cells of Saccharomyces cerevisiae strain IFFI1051 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 35 MHz at 26 mV/cm for 12 hours; 39 MHz at 26 mV/cm for 12hours; 43 MHz at 26 mV/cm for 12 hours; 47 MHz at 26 mV/cm for 12 hours;35 MHz at 150 mV/cm for 24 hours; 39 MHz at 150 mV/cm for 24 hours; 43MHz at 150 mV/cm for 24 hours, 47 MHz at 150 V/cm for 24 hours. Thenumber of Bacillus anthracis in a sample was reduced by, more than 3.1%relative to the control with no yeasts. There was no significant changein the number of pathogens in the control containing non-activatedcells.

[0184] Using cells of Saccharomyces cerevisiae strain IFFI1331 which hadbeen cultured in the presence of a series of 8 EM fields in the order,stated: 33 MHz at 26 mV/cm for 12 hours; 36 MHz at 26 mV/cm for 12hours; 45 MHz at 26 mV/cm for 12 hours; 47 MHz at 26 mV/cm for 12 hours;33 MHz at 150 mV/cm for 24 hours; 36 MHz at 150 mV/cm for 24 hours; 45MHz at 150 mV/cm for 24 hours; 47 MHz at 150 mV/cm for 24 hours. Thenumber of Mycobacterium tuberculosis in a sample was reduced by morethan 2.9% relative to the control with no yeasts. There was nosignificant change in the number of pathogens in the control containingnon-activated cells.

[0185] Using cells of Saccharomyces cerevisiae strain IFFI1345 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 30 MHz at 26 mV/cm for 12 hours; 34 MHz at 26 mV/cm for 12hours; 38 MHz at 26 mV/cm for 12 hours; 49 MHz at 26 mV/cm for 12 hours;30 MHz at 150 mV/cm for 24 hours; 34 MHz at 150 mV/cm for 24 hours; 38MHz at 150 mV/cm for 24 hours; 49 MHz at 150 mV/cm for 24 hours. Thenumber of Escherichia coli in a sample was reduced by more than 48%relative to the control with no yeasts. There was no significant changein the number of pathogens in the control containing non-activatedcells.

[0186] Using cells of Saccharomyces cerevisiae strain IFFI1211 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 30 MHz at 26 mV/cm for 12 hours; 33 MHz at 26 mV/cm for 12hours; 36 MHz at 26 mV/cm for 12 hours; 38 MHz at 26 mV/cm for 12 hours;30 MHz at 150 mV/cm for 24 hours; 33 MHz at 150 mV/cm for 24 hours; 36MHz at 150 mV/cm for 24 hours; 38 MHz at 150 mV/cm for 24 hours. Thenumber of Salmonella species bacteria in a sample was reduced by morethan 66% relative to the control with no yeasts. There was nosignificant change in the number of pathogens in the control containingnon-activated cells.

[0187] 5.9. Yeast Cell Component that Decomposes Undesirable Chemicals

[0188] The present invention further provides yeast cells that arecapable of degrading undesirable chemicals, such as antibiotics, thatare typically found in manure

[0189] According to the invention, the ability of yeasts to degradeantibiotics is activated or enhanced by culturing the yeasts in thepresence of an electromagnetic field. The resulting yeast cells can beused as a component in the biological fertilizer compositions of theinvention.

[0190] The frequency of the electromagnetic field for activating orenhancing the ability of yeasts to degrade undesirable chemicals, inparticular antibiotics, can generally be found in the range of 70 MHz to100 MHz. After sufficient time is given for the yeast cells to grow, theyeast cells can be tested for their enhanced ability to decomposeantibiotics by methods well known in the art. Antibiotics degraded bythe yeasts of the invention include but are not limited to moleculeswithin the families of beta-lactams, tetracyclines, polypeptides,glycopeptides, aminoglycosides, and macrolides.

[0191] The method of the invention for making antibiotics-degradingyeasts is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0192] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃,MgSO₄, NaCl, and CaSO₄. TABLE 9 Composition for a culture medium foryeasts that degrade undesirable chemicals Medium Composition QuantitySoluble Starch 8.0 g, > 120 mesh Sucrose 5 g NaCl 0.2 g MgSO₄.7H₂O 0.2 gCaCO₃.5H₂O 0.5 g CaSO₄.2H₂O 0.2 g Peptone 15 g K₂HPO₄ 0.5 g Autoclavedwater 1000 ml Sludge extract 600 ml

[0193] The sludge extract for the culture medium is prepared byincubating 500 g of fresh sludge in about 600 ml of warm water (at35-40° C.) for 24 hours at 30-37° C., and filtering the fluid to removeparticulate matters. It should be noted that the composition of themedia provided in Table 9 is not intended to be limiting. Variousmodifications of the culture medium may be made by those skilled in theart, in view of practical and economic considerations, such as the scaleof culture and local supply of media components.

[0194] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0195] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of 70.000 to 100.000 MHz.For example and without being limited by such examples, each EM fieldcan have a frequency at about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 100 MHz. The field strength of the EM field(s) is in the range of40 to 250 mV/cm. If a series of EM fields are applied, the EM fields caneach have a different frequency within the stated range, or a differentfield strength within the stated range, or different frequency and fieldstrength within the stated ranges. In a preferred embodiment, the EMfield(s) at the beginning of a series have a lower EM field strengththan later EM field(s), such that the yeast cell culture are exposed toEM fields of progressively increasing field strength. Although anypractical number of EM fields can be used within a series, it ispreferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6,7, or 8 different EM fields in a series.

[0196] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e.g., one or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EMs field or EM fields for a total of about 180-328hours.

[0197] For example, using an. exemplary apparatus as depicted in FIG. 1,an initial field strength in the range of 40-60 mV/cm, usually at about50 mV is used. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions for anotherperiod, except that the amplitude is increased to a higher level in therange of 100-250 mV/cm, usually to about 200 mV/cm. The process of theinvention is carried out at temperatures ranging from about 23° to 30°C.; however, it is preferable to conduct the process at 25° to 28° C.The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

[0198] At the end of the culturing process, the yeast cells may berecovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C. The recovered yeastcells may also be dried and stored in powder.

[0199] To determine the activity of the activated yeast cells towards anantibiotic compound, methods well known in the art, such as HPLC, can beused to measure the amounts of the antibiotic compound in a test sampleat various time point and under different incubation conditions. Forexample, a known amount of an antibiotic (up to 100 mg per liter) isadded to 10 liter of an aqueous extract of the manure. Then, 0.1 ml eachof activated and non-activated yeasts (at least 10⁷ cells/ml) are addedto the 10 liter samples containing the antibiotics, and incubated for 24hours at 28° C. A control is also included which does not contain anyyeast cells. After 24 hours, the amounts of antibiotics remaining in theextracts are determined and compared by performing HPLC on samples ofthe extracts.

[0200] Using cells of Saccharomyces cerevisiae strain AS2.293 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 77 MHz at 48 mV/cm for 15 hours; 83 MHz at 48 mV/cm for 15hours; 90 MHz at 48 mV/cm for 15 hours; 96 MHz at 48 mV/cm for 15 hours;77 MHz at 200 mV/cm for 30 hours; 83 MHz at 200 mV/cm for 30 hours; 90MHz at 200 mV/cm for 30 hours; 96 MHz at 200 mV/cm for 30 hours. Theamount of penicillin G in a sample was reduced by more than 23% relativeto the control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

[0201] Using cells of Saccharomyces cerevisiae strain IFFI1063 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 70 MHz at 48 mV/cm for 15 hours; 73 MHz at 48 mV/cm for 15hours; 88 MHz at 48 mV/cm for 15 hours; 98 MHz at 48 mV/cm for 15 hours;70 MHz at 200 mV/cm for 30 hours; 73 MHz at 200 mV/cmn for 30 hours; 88MHz at 200 mV/cm for 30 hours; 98 MHz at 200 mV/cm for 30 hours. Theamount of chlorotetracycline in a sample was reduced by more than 31%relative to the control with no yeasts. There was no significant changein the concentration of the antibiotic in the control containingnon-activated cells.

[0202] Using cells of Saccharomyces cerevisiae strain IFFI1221 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 70 MHz at 48 mV/cm for 15 hours; 74 MHz at 48 mV/cm for 15hours; 88 MHz at 48 mV/cm for 15 hours; 98 MHz at 48 mV/cm for 15 hours;70 MHz at 200 mV/cm for 30 hours; 74 MHz at 200 mV/cm for 30 hours; 88MHz at 200 mV/cm for 30 hours; 98 MHz at 200 mV/cm for 30 hours. Theamount of oxytetracycline in a sample was reduced by more than 28%relative to the control with no yeasts. There was no significant changein the concentration of the antibiotic in the control containingnon-activated cells.

[0203] Using cells of Saccharomyces cerevisiae strain IFFI1340 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 71 MHz at 48 mV/cm for 15 hours; 73 MHz at 48 mV/cm for 15hours; 77 MHz at 48 mV/cm for 15 hours; 88 MHz at 48 mV/cm for 15 hours;71 MHz at 200 mV/cm for 30 hours; 73 MHz at 200 mV/cm for 30 hours; 77MHz at 200 mV/cm for 30 hours; 88 MHz at 200 mV/cm for 30 hours. Theamount of doxycycline in a sample was reduced by more than 33% relativeto the control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

[0204] Using cells of Saccharomyces cerevisiae strain IFFI1215 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 70 MHz at 48 mV/cm for 15 hours; 75 MHz at 48 mV/cm for 15hours; 82 MHz at 48 mV/cm for 15 hours; 85 MHz at 48 mV/cm for 15 hours;70 MHz at 200 mV/cm for 30 hours; 75 MHz at 200 mV/cm for 30 hours; 82MHz at 200 mV/cm for 30 hours; 85 MHz at 200 mV/cm for 30 hours. Theamount of tetracycline in a sample was reduced by more than 26% relativeto the control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

[0205] Using cells of Saccharomyces cerevisiae strain IFFI1213 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 70 MHz at 48 mV/cm for 15 hours; 73 MHz at 48 mV/cm for 15hours; 80 MHz at 48 mV/cm for 15 hours; 96 MHz at 48 mV/cm for 15 hours;70 MHz at 200 mV/cm for 30 hours; 73 MHz at 200 mV/cm for 30 hours; 80MHz at 200 mV/cm for 30 hours; 96 MHz at 200 mV/cm for 30 hours. Theamount of streptomycin in a sample was reduced by more than 31% relativeto the control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

[0206] Using cells of Saccharomyces cerevisiae strain IFFI106 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 71 MHz at 48 mV/cm for 15 hours; 78 MHz at 48 mV/cm for 15hours: 86 MHz at 48 mV/cm for 15 hours; 98 MHz at 48 mV/cm for 15 hours;71 MHz at 200 mV/cm for 30 hours; 78 MHz at 200 mV/cm for 30 hours; 86MHz at 200 mV/cm for 30 hours; 98 MHz at 200 mV/cm for 30 hours. Theamount of kanamycin in a sample was reduced by more than 25% relative tothe control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

[0207] Using cells of Saccharomyces cerevisiae strain IFFI1211 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 73 MHz at 48 mV/cm for 15 hours; 79 MHz at 48 mV/cm for 15hours; 88 MHz at 48 mV/cm for 15 hours; 98 MHz at 48 mV/cm for 15 hours;73 MHz at 200 mV/cm for 30 hours; 79 MHz at 200 mV/cm for 30 hours; 88MHz at 200 mV/cm for 30 hours; 98 MHz at 200 mV/cm for 30 hours. Theamount of erythromycin in a sample was reduced by more than 27% relativeto the control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

[0208] Using cells of Saccharomyces cerevisiae strain IFFI210 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 70 MHz at 48 mV/cm for 15 hours; 77 MHz at 48 mV/cm for 15hours; 84 MHz at 48 mV/cm for 15 hours; 93 MHz at 48 mV/cm for 15 hours;70 MHz at 200 mV/cm for 30 hours; 77 MHz at 200 mV/cm for 30 hours; 84MHz at 200 mV/cm for 30 hours; 93 MHz at 200 mV/cm for 30 hours. Theamount of spiramycin in a sample was reduced by more than 22% relativeto the control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

[0209] Using cells of Saccharomyces cerevisiae strain IFFI1260 which hadbeen cultured in the presence of a series of 8 EM fields in the orderstated: 75 MHz at 48 mV/cm for 15 hours; 78 MHz at 48 mV/cm for 15hours; 81 MHz at 48 mV/cm for 15 hours; 95 MHz at 48 mV/cm for 15 hours;75 MHz at 200 mV/cm for 30 hours; 78 MHz at 200 mV/cm for 30 hours; 81MHz at 200 mV/cm for 30 hours; 95 MHz at 200 mV/cm for 30 hours. Theamount of bacitracin in a sample was reduced by more than 17% relativeto the control with no yeasts. There was no significant change in theconcentration of the antibiotic in the control containing non-activatedcells.

[0210] 5.10. Odor-reducing Yeast Cell Component

[0211] The present invention also provides yeast cells that are capableof reducing the odor of Sludge. Without being bound by any theory, theinventor believes that the yeast cells of the invention are capable ofreducing the odor of sludge by modifying or decomposing known andunknown compounds in the manure that are malodorous. However, it is notnecessary to demonstrate that such compounds have been decomposed. It issufficient so long as the odor is reduced as determined subjectively bya panel of subjects, after the yeast cells of the invention have beenused.

[0212] According to the present invention, yeast cells that are capableof reducing the odor of organic materials are prepared by culturing thecells in the presence of an electromagnetic field in an appropriateculture medium. The frequency of the electromagnetic field foractivating or enhancing this ability in yeasts can generally be found inthe range of 2160 to 2380 MHz. After sufficient time is given for theyeast cells to grow, the yeast cells can be tested for their ability toreduce the odor of organic materials by methods well known in the art.

[0213] The method of the invention for making the odor-reducing yeastcells is carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

[0214] Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are (NH₄)₂HPO₄, K₂HPO₄, CaCO₃,MgSO₄, NaCl, and CaSO₄. TABLE 10 Composition for a culture medium foryeasts that reduce odor Medium Composition Quantity Sludge 100 g NaCl0.2 g MgSO₄.7H₂O 0.2 g CaCO₃.5H₂O 0.5 g CaSO₄.2H₂O 0.2 g K₂HPO₄ 0.5 gAutoclaved water 900 ml

[0215] It should be noted that the composition of the media provided inTable 10 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

[0216] The process can be initiated by inoculating 100 ml of medium with1 ml of an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown in thepresence of an electromagnetic (EM) field, or a series of EM fields. Ifa series of EM fields are applied, the yeast culture can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

[0217] The EM field(s), which can be applied by any means known in theart, can each have a frequency in the range of 2160.000 to 2380.000 MHz,and preferably in the ranges of 2160 to 2250 MHz or 2280 to 2380 MHz.For example and without being limited by such examples, each EM fieldcan have a frequency at about 2160, 2165, 2170, 2175, 2180, 2185, 2190,2195, 2200, 2205, 2210, 2215, 2220, 2225, 2230, 2235, 2240, 2245, 2250,2280, 2285, 2290, 2295, 2300, 2305, 2315, 2320, 2325, 2330, 2335, 2340,2345, 2350, 2355, 2360, 2365, 2370, 2375, or 2380 MHz. The fieldstrength of the EM field(s) is in the range of 0.5 to 320 mV/cm,preferably 30 to 310 mV/cm. If a series of EM fields are applied, the EMfields can each have a different frequency within the stated range, or adifferent field strength within the stated range, or different frequencyand field strength within the stated ranges. In a preferred embodiment,the EM field(s) at the beginning of a series have a lower EM fieldstrength than later EM field(s), such that the yeast cell culture areexposed to EM fields of progressively increasing field strength.Although any practical number of EM fields can be used within a series,it is preferred that the yeast culture be exposed to a total of 2, 3, 4,5, 6, 7, or 8 different EM fields in a series.

[0218] Although the yeast cells will become activated even after a fewhours of culturing in the presence of the EM field(s), and the Yeastcells can be cultured in the presence of the EM field(s) for an extendedperiod of time (e g., two or more weeks), it is generally preferred thatthe activated yeast cells be allowed to multiply and grow in thepresence of the EM field or EM fields for a total of about 80-320 hours.

[0219] The process of the invention is carried out at temperaturesranging from about 23° to 30° C.; however, it is preferable to conductthe process at 25 to 28° C. The culturing process may preferably beconducted under conditions in which the concentration of dissolvedoxygen is between 0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygenlevel can be controlled by any conventional means known to one skilledin the art, including but not limited to stirring and/or bubbling.

[0220] At the end of the culturing process, the yeast cells may berecovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The recovered yeast cellsmay also be dried and stored in powder form.

[0221] Any methods known in the art can be used to test the culturedyeast cells for their ability to reduce the odor of organic materials.The amount of malodorous chemicals such as hydrogen sulfide, ammonia,indole, p-cresol, skatol, and organic acids present in a test sample oforganic material can be determined by any methods known in the art,including but not limited to gas phase chromatography, olfactometry,mass spectrometry, or the use of an odor panel.

[0222] To determine the activity of the activated yeast cells towards anmalodorous compound, methods well known in the art, such as HPLC or massspectrometry (e.g., VG micromass), can be used to measure the amounts ofthe malodorous compound in a test sample at various time point and underdifferent incubation conditions. For example, a known amount of amalodorous compound (up to 100 mg per liter) is added to 10 liter of anaqueous extract of manure. Then, 0.1 ml of activated and non-activatedyeasts (at least 10⁷ cells/ml) are added to the 10 liter samplescontaining the antibiotics, and incubated for 24 hours at 28° C. Acontrol is also included which does not contain any yeast cells. After24 hours, the amounts of the malodorous compounds remaining in theextracts are determined and compared.

[0223] Accordingly, the odor caused by hydrogen sulfide and otherrelated sulfur-containing or sulfhydryl (SH-) containing molecules canbe reduced by yeasts cultured in the presence of an EM field that is inthe range of 2160.000 to 2250.000. Using cells of Saccharomycescerevisiae strain AS2.559 which had been cultured in the presence of aseries of four EM fields in the order stated: 2165 MHz at 240 mV/cm for20 hours; 2175 MHz at 240 mV/cm for 20 hours; 2200 MHz at 240 mV/cm for20 hours; and 2235 MHz at 240 mV/cm for 20 hours, the amount of hydrogensulfide in a sample was reduced by more than 13% relative to the controlcontaining no yeasts. There was no significant reduction in themalodorous compound in the sample containing non-activated yeasts

[0224] The odor caused by ammonia and related NH-containing compoundscan be reduced by yeasts cultured in the presence of an EM field that isin the range of 2160.000 to 2250.000. Using cells of Saccharomycescerevisiae strain AS2.423 which had been cultured in the presence of aseries of four EM fields in the order stated: 2160 MHz at 250 mV/cm for20 hours; 2175 MHz at 250 mV/cm for 20 hours; 2210 MHz at 250 mV/cm for20 hours; and 2245 MHz at 250 mV/cm for 10 hours, the amount of ammoniain a sample was reduced by more than 11% relative to the controlcontaining no yeasts. There was no significant reduction in themalodorous compound in the sample containing non-activated yeasts.

[0225] The odor caused by indole and other related molecules, such asskatol, can be reduced by yeasts cultured in the presence of an EM fieldthat is in the range of 2160.000 to 2250.000. Using cells ofSaccharomyces cerevisiae strain AS2.612 which had been cultured in thepresence of a series of four EM fields in the order stated: 2165 MHz at240 mV/cm for 40 hours; 2180 MHz at 240 mV/cm for 20 hours; 2200 MHz at240 mV/cm for 40 hours; and 2220 MHz at 240 mV/cm for 20 hours, theamount of indole in a sample was reduced by more than 15% relative tothe control containing no yeasts. There was no significant reduction inthe malodorous compound in the sample containing non-activated yeasts.

[0226] The odor caused by organic acids (such as formic acid, aceticacid, propanoic acid, butyric acid, and other volatile fatty acids) canbe reduced by yeasts cultured in the presence of an EM field that is inthe range of 2280.000 to 2380.000. Using cells of Saccharomycescerevisiae strain AS2.53 which had been cultured in the presence of aseries of four EM fields in the order stated: 2315 MHz at 290 mV/cm for30 hours; 2335 MHz at 290 mV/cm for 10 hours; 2355 MHz at 290 mV/cm for20 hours; and 2375 MHz at 290 mV/cm for 10 hours, the amount of aceticacid in a sample was reduced by more than 19% relative to the controlcontaining no yeasts. There was no significant reduction in themalodorous compound in the sample containing non-activated yeasts.

[0227] The odor caused by methylamine, dimethylamine, trimethylamine,and other aliphatic substituted amines can be reduced by yeasts culturedin the presence of an EM field that is in the range of 2160.000 to2250.000. Using cells of Saccharomyces cerevisiae strain AS2.541 whichhad been cultured in the presence of a series of four EM fields in theorder stated: 2160 MHz at 250 mV/cm for 20 hours; 2190 MHz at 250 mV/cmfor 10 hours; 2210 MHz at 250 mV/cm for 40 hours; and 2250 MHz at 250mV/cm for 40 hours, the amount of methyl-substituted amides in a samplewas reduced by more than 23% relative o the control containing noyeasts. There was no significant reduction in the malodorous compound inthe sample containing non-activated yeasts.

[0228] The odor caused by p-cresol and related compounds can be reducedby yeasts cultured in the presence of an EM field that is in the rangeof 2280.000 to 22380.000. Using cells of Saccharomyces cerevisiae strainAS2.163 which had been cultured in the presence of a series of four EMfields in the order stated: 2300, MHz at 98 mV/cm for 20 hours; 2370 MHzat 98 mV/cm for 15 hours; 2300 MHz at 250 mV/cm for 20 hours; and 2370MHz at 250 mV/cm for 30 hours, the amount of p-cresol in a sample wasreduced by more than 23% relative to the control containing no yeasts.There was no significant reduction in the malodorous compound in thesample containing non-activated yeasts.

[0229] 5.11. Formation of Symbiosis-like Relationships

[0230] In another embodiment of the present invention, yeast cells withthe newly activated or enhanced ability to (1) fix nitrogen, (2)decompose phosphorus-containing minerals or compounds, (3) balancephosphorus compounds, (4) decompose insoluble potassium-containingminerals or compounds, and (5) decompose complex carbon compounds asdescribed in Sections 5.1-5.5 are combined and cultured so that theyform a symbiosis-like relationship whereby they can grow togetherwithout substantially relying on outside supplies of biologicalavailable nitrogen, phosphorus, potassium, and carbon nutrients. Thenutrients needed for growth are supplied by the respectivenutrient-producing yeast strain within the fertilizer composition byconverting biologically-unavailable nutrients from various sources intoavailable nutrients. The activity of each of the yeast strains inproducing the respective types of nutrient relates in part to the needsof other yeast cells as well as the plants. As a result, soluble,biologically-available nutrients will be converted when needed, therebyavoiding excess losses due to, for example, leaching.

[0231] The optional process which can be used to improve the performanceof the biological fertilizer is described as follows. At least fourstrains of yeasts prepared according to Sections 5.1-5.5 are mixed andcultured in the presence of an electromagnetic field in an appropriateliquid medium. The medium contains nitrogen, phosphorus, potassium, andcarbon nutrients in biologically unavailable forms. As non-limitingexamples, atmospheric nitrogen is used as the source of nitrogennutrient, powder of phosphate rock is used as the source of phosphorusnutrient, powder of potassium mica is used as the source of potassiumnutrient, and powdered cellulose is used as the source of complex carbonnutrient. Other forms of insoluble phosphorus and potassium-containingsubstances and complex carbon compounds may also be used in place of orin combination with any of the above-identified minerals as sources ofphosphorus, potassium, and carbon nutrients. Among the inorganic saltswhich can be incorporated in the culture media are the customary saltscapable of yielding sodium, calcium, sulfate, carbonate, and like ions.Non-limiting examples of nutrient inorganic salts are CaCO₃, MgSO₄,NaCl, and CaSO₄. TABLE 11 Composition for a culture medium for formationof symbiosis-like relation Medium Composition Quantity NaCl 0.5 gMgSO₄.7H₂O 0.4 g CaCO₃.5H₂O 3.0 g CaSO₄.2H₂O 0.3 g Yeast extract paste0.3 g Potassium mica 1.2 g; Powder of > 200 mesh Rock phosphate 1.2 g;Powder of > 200 mesh Cellulose 5.0 g; Powder of > 200 mesh Autoclavedwater 1000 ml

[0232] It should be noted that the composition of the media provided inTable 11 is not intended to be limiting. Various modifications of theculture medium may be made by those skilled in the art, in view ofpractical and economic considerations, such as the scale of culture andlocal supply of media components.

[0233] The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling. The process ofthe invention is carried out at temperatures ranging from about 25° to30° C.; however, it is preferable to conduct the process at 28° C. Theprocess is initiated in sterilized medium by inoculating typically about20 ml of each inoculum of the four strains of yeast cells, each at acell density of about 10⁸ cell/ml. The optional process can be scaled upor down according to needs.

[0234] The yeast culture is grown for 12-72 hours, preferably for about48 hours, in the presence of four independent electromagnetic fields.The electromagnetic fields, which car be applied by a variety of means,each has the following respective frequencies: (1) in the range of about840 to about 916 MHz for nitrogen-fixing; (2) in the range of about 300to about 500 MHz for phosphorus-decomposing or phosphorus balancing; (3)in the range of about 100 to about 300 MHz for potassium-decomposing;and (4) in the range of about 1000 to about 1200 MHz for complexcarbon-decomposing. Generally, the yeast cells are subjected to an EMfield strength in the range from, 5 mV/cm to 160 mV/cm per completecycle. Using an exemplary apparatus as depicted in FIG. 2, the outputamplitude of the EM waves used are in the range of 0-3 mV, preferably20-1800 mV. The amplitude of each electromagnetic field is repeatedlycycled between 0 mV to 3000 mV, preferably between 20 mV to 1800 mV, insteps of 1 mV at a rate of about two to about ten minutes per completecycle.

[0235] 5.12. Soil Adaptation

[0236] The yeast cells of the invention must also be able to grow andperform their respective functions in various types of soils. Theability of the yeast cells to survive and grow can be enhanced byadapting the yeast cells of the invention to a particular soilcondition.

[0237] In another embodiment of the invention, yeast cells preparedaccording to any one of Sections 5.1-5.10 can be cultured separately orin a mixture in a solid or semi-solid medium containing soil from one ormore soil sources. This optional process which can be used to improvethe performance of the biological fertilizer described by way of anexample as follows.

[0238] A suspension containing 10 ml of yeasts at a density of 10⁶cell/ml is mixed with a 1000 cm³ of the soil medium. The process can bescaled up or down according to needs. The mixture of yeast and soil iscultured for about 48-96 hours, preferably for about 48 hours, in thepresence of an electromagnetic field. The electromagnetic field, whichcan be applied by a variety of means, has a frequency that, depending onthe function of the yeasts, corresponds to one of the frequenciesdescribed in Sections 5.1-5.10. Generally, the yeast cells are subjectedto an EM field strength in the range from 60 mV/cm to 250 mV/cm in thisprocess.

[0239] The culture is incubated at temperatures that cycle between about3° C. to about 48° C. For example, in a typical cycle, the temperatureof the culture may start at 35-48° C. and be kept at this temperaturefor about 1-2 hours, then adjusted up to 42-45° C. and kept at thistemperature for 1-2 hours, then adjusted to 26-30° C. and kept at thistemperature for about 2-4 hours, and then brought down to 5-10° C. andkept at this temperature for about 1-2 hours, and then the temperaturemay be raised again to 35-45° C. for another cycle. The cycles arerepeated until the process is completed. After the last temperaturecycle is completed, the temperature of the culture is lowered to 3-4° C.and kept at this temperature for about 5-6 hours. After adaptation, theyeast cells may be isolated and recovered from the medium byconventional methods, such as filtration. The adapted yeast cells can bestored under 4° C. An exemplary set-up of the culture process isdepicted in FIG. 3.

[0240] 5.13. Separation or Enrichment of Yeast Cells

[0241] Yeast cells that have been adapted to form a symbiosis-likerelationship according to Section 5.11 can be separated or enriched insuch a way that each strain of yeast cells keep their acquired orenhanced functions. Separation of yeast cells is carried out accordingto methods described in U.S. Pat. No. 5,578,486 and Chinese patentpublication CN 1110317A which are incorporated herein by reference inits entirety. The same frequency used for activating the yeast cells maybe used during the separation process. The separated yeast cells canthen be dried, and stored.

[0242] 5.14. Manufacture of the Biological Fertilizers

[0243] In addition to the yeast cell components, sludge and optionallyinorganic materials are also included in the biological fertilizercompositions of the invention. The preparation of manure and suchmaterials as well as the steps involved in the manufacture of thebiological fertilizer compositions are described below.

[0244] 5.14.1. Preparation of the Organic and Inorganic SubstrateComponents

[0245] Sludge from many varied sources can be used in the biologicalfertilizer compositions of the present invention. Mixtures of sludgefrom different sources can also be used. Organic compounds present inthe sludge are decomposed by the yeasts of the invention. Depending onthe source of the sludge, in addition to nitrogen, it may contain anuseful amount of phosphorus (e.g., P₂O₅) and potassium (e.g., K₂O).Nutrient concentrations in sludge, can vary due to its origin,processing if any, methods of storage and moisture content. Methodsknown in the art can be employed to determine the nutrient value of eachbatch of sludge prior to its use in making a biological fertilizercomposition.

[0246] Inorganic materials, such as but not limited to phosphate rockand potassium mica, can optionally be included as additional sources ofphosphorus and potassium respectively. Other phosphorous- orpotassium-containing materials and minerals can also be used. Theseinorganic compounds are decomposed by K-decomposing and P-decomposingyeast cells into biologically available potassium and biologicallyavailable phosphorus that can be used by the growing plants as well asthe yeast cells in the fertilizer.

[0247] Any inorganic material may be used in combination with sludge inthe present invention. Alternatively, the inorganic ingredients may beomitted, or substituted by another if it is deemed desirable by theparticular application. For example, phosphate rock can be omitted ifsludge is used which contains sufficient biologically availablephosphorus.

[0248] The sludge is preferably dried to a moisture content of ≦5%. Boththe dried sludge and optional inorganic substrate components in thepresent invention are ground into suitable forms and sizes beforeincorporated into the fertilizer. Typically, the sludge or inorganicmaterial is conveyed into a crusher where it is broken up into pieces of≦5 cm in diameter. Any conventional crusher or equivalent machines canbe used for this purpose. The pieces are then transferred to a grinderby any conveying means and ground to a powder of ≧150 mesh. Any grinderthat allows fine grinding can be used for this purpose. The powder isthen conveyed to an appropriate storage tank for storage until use withother components of the fertilizer. A schematic illustration of thegrinding process is shown in FIGS. 4 and 5.

[0249] 5.14.2. Fermentation Process Using Growth Factor-Producing Yeast

[0250] In the present invention, the preparation of GF-producing yeastis carried out in a fermentation process using as seed the activatedyeast strain as described in Section 5.6. A schematic of thefermentation process is illustrated in FIG. 6.

[0251] The fermentation medium is prepared according to a ratio of 2.5liters of water per kilogram of starch. Clean water, preferably waterfree of any microorganisms, is used to prepare the fermentation medium.The fermentation is carried out at a temperature between 20-30° C.,preferably between 25-28° C., in a clean environment and in a spacewhere there are no strong sources of electromagnetic fields, such aspower lines and power generators. Any equipments that contact thefermentation broth, including reactors, pipelines, and stirrers, must bethroughly cleaned before each use. The fermentation process normallylasts about 48-72 hours at 28-30° C. when at least 90% of thefermentation substrate is fermented. Fermentation is preferablyconducted under semi-aerobic conditions or conditions in which theoxygen level is about 20-60% of the maximal soluble oxygenconcentration. The oxygen level can be controlled by any conventionalmeans known to one skilled in the art including but not limited tostirring and/or bubbling. After fermentation, the cell counts shouldreach about 2×10¹⁰ cells/ml. The fermentation broth is kept at atemperature in the range of 15-28° C. and must be used within 24 hours.Alternatively, the GF-producing yeasts an be drained, dried and storedin powder form.

[0252] 5.14.3. Fermentation Process Using ATP-Producing Yeast

[0253] In the present invention, the preparation of ATP-producing yeastis carried out by a fermentation process using as seed the adapted yeaststrain as described in Section 5.7. A schematic of the fermentationprocess is illustrated in FIG. 6.

[0254] The fermentation medium is prepared according to a ratio of 2.5liters of water per kilogram of starch. Clean water, preferably waterfree of any microorganisms, most preferably autoclaved water, is used toprepare the fermentation media. The fermentation is carried out at atemperature between 20-30° C., preferably between 25-28° C., in a cleanenvironment and in a space where there are no strong sources ofelectromagnetic fields, such as power lines and power generators. Anyequipments that contact the fermentation broth, including reactors,pipelines, and stirrers, must be throughly cleaned before each use. Thefermentation process normally lasts about 48-72 hours, depending on thefermentation temperature. Preferably at the end of the process at least90% of the fermentation substrate is fermented. Fermentation ispreferably conducted under semi-aerobic conditions or conditions inwhich the oxygen level is about 20-60% of the maximal soluble oxygenconcentration. The oxygen level can be controlled by any conventionalmeans known to one skilled in the art, including but not limited tostirring and/or bubbling. After fermentation, the cell counts shouldreach about 2×10¹⁰ cells/ml. The fermentation broth is kept at atemperature in the range of 15-28° C. and must be used within 24 hours.Alternatively, the ATP-producing yeasts can be drained, dried and storedin powder form.

[0255] 5.14.4. Preparation of Mixture of Raw Materials

[0256] Sludge and the optional inorganic raw materials are mixed inexemplary proportions as shown in Table 12. Appropriate amount oforganic and inorganic materials prepared according to Section 5.10.1 andstarch are conveyed to a mixer. Any conventional mixer, such as but notlimited a rotary drum mixer, can be used. The mixing tank is rotatedconstantly so that powders of sludge and starch are mixed evenly. Themixture is then conveyed to a storage tank. The procedure for mixingsludge and inorganic substrate material is illustrated in FIG. 7. TABLE12 Ratio of raw materials Material Percentage Requirement Powder ofdried sludge 58-63% ≧ 150 mesh, water content ≦ 5% Powder of inorganicmaterials   20% ≧ 150 mesh, water content ≦ 3% Starch 10-15% regularstarch powder, water content ≦ 8%

[0257] 5.14.5. Preparation of Yeast Mixture

[0258] If no inorganic materials is used, the proportion of sludge canbe increased up to 80%. A yeast mixture is prepared in the exemplaryproportions as shown in Table 13. Appropriate amounts of the nine yeaststrains in dried powder form prepared according to Section 5.1-5.10 areconveyed to a mixing tank. The yeasts are allowed to mix for about 10-20minutes. The mixture is then transferred to a storage tank. Anyequipments used for mixing yeasts, including the mixing tank and thestorage tank, must be throughly cleaned, preferably sterilized, beforeeach use. The yeast mixture is stored at a temperature below 20° C. andmust be used within 24 hours. The procedure for mixing yeasts isillustrated in FIG. 8. Alternatively, the mixture of nine yeasts can bedried and stored in powder form. TABLE 13 Ratio of microorganismsPercentage Yeast Quantity (dry weight) Note Nitrogen-fixing yeast1.0-2.0 kg 0.1-0.2% Dry yeast powder Phosphorus- 1.0-2.0 kg 0.1-0.2% Dryyeast powder decomposing yeast Potassium- 1.0-2.0 kg 0.1-0.2% Dry yeastpowder decomposing yeast Carbon-decomposing 1.0-2.0 kg 0.1-0.2% Dryyeast powder yeast Pathogen-suppressing 1.0-2.0 kg 0.1-0.2% Dry yeastpowder yeast Chemical- 1.0-2.0 kg 0.1-0.2% Dry yeast powder decomposingyeast Odor-reducing yeast 1.0-2.0 kg 0.1-0.2% Dry yeast powder Growthfactor- 25 L 1% Yeast fermentation producing yeast broth ATP-producingyeast 75 L 3% Yeast fermentation broth

[0259] 5.14.6. Manufacture of Biological Fertilizer

[0260] The biological fertilizer of the present invention is produced bymixing the yeast mixture of Section 5.14.5 and the mixture of theorganic and inorganic materials of Section 5.14.1 at a ratio accordingto Table 14. For example, the yeasts and the sludge, and inorganicmaterials are conveyed to a granulizer to form granules. The granules ofthe fertilizer are then dried in a two-stage drying process. During thefirst drying stage, the fertilizer is dried in a first dryer at atemperature not exceeding 65° C. for a period of time not exceeding 10minutes so that yeast cells quickly become dormant. The fertilizer isthen send to a second dryer and dried at a temperature not exceeding 70°C. for a period of time not exceeding 30 minutes to further removewater. After the two stages, the water content should be lower than 5%.It is preferred that the temperatures and drying times be adhered to inboth drying stages so that yeast cells do not lose their vitality andfunctions. The fertilizer is then cooled to room temperature. Thefertilizer may also be screened in a separator so that fertilizergranules of a preferred size are selected. Any separator, such as butnot limited to a turbo separator with adjustable speed and screen sizes,can be used. The fertilizer of the selected size is then sent to a bulkbag filler for packing.

[0261] The production process is illustrated in FIGS. 9-11. FIG. 9 is aschematic illustration of the procedure for producing the fertilizerfrom its components. FIG. 10 is a schematic illustration of the dryingprocess. FIG. 11 is a schematic illustration of the cooling and packingprocess. TABLE 14 Composition of the biological fertilizer (for onemetric ton of fertilizer) Percentage (dry Quantity weight) Note Mixtureof raw materials 952-956 kg 95.2-95.4% Dry weight Mixture of yeasts 100L 4.4-4.8% Dry weight

6. EXAMPLE

[0262] The following examples demonstrate the manufacture of anexemplary biological fertilizer composition of the present invention.These examples represent a preferred embodiment of the presentinvention.

[0263] 6.1 Biological Fertilizer Composition Comprising Sludge

[0264]Saccharomyces cerevisiae strains having accession numbers AS2.628,AS2.631, AS2.982, AS2.413 and AS2.536 are used to prepare the yeast cellcomponents of the biological fertilizer composition. All were depositedin China General Microbiological Culture Collection Center (CGMCC),China Committee for Culture Collection of Microorganisms. Yeast strainAS2.628 is cultured according to the method described in Section 5.1 fornitrogen-fixation; and yeast strain AS2.399 to the method described inSection 5.2 for P-decomposition. Yeast strain AS2.631 is culturedaccording to the method described in Section 5.4 for K-decomposition.Yeast strain AS2.982 is cultured according to the method described inSection 5.5 for C-decomposition. Yeast strain AS2.413 is culturedaccording to the method described in Section 5.6 for production ofgrowth factor. Yeast strain AS2.536 is cultured according to the methoddescribed in Section 5.7 for ATP production. Yeast strain IFFI1301 iscultured according to the method described in Section 5.8 forsuppressing growth of pathogens. Yeast strain IFFI1291 is culturedaccording to the method described in Section 5.9 for degradingundesirable chemicals. Yeast strain IFFI1202 is cultured according tothe method described in Section 5.10 for odor reduction.

[0265] Dried sludge in powder form was prepared as described in Section5.14.5.

[0266] The production of growth factor-producing yeast is carried out ina fermentation process using as seed the activated yeast strain AS2.413as described in Section 5.6. A schematic of the fermentation process isillustrated in FIG. 6. The fermentation medium is prepared according toa ratio of 2.5 liters of clean water per kilogram of starch and 10kilograms of starch per metric ton of biological fertilizer. Thefermentation medium is inoculated according to a ratio of 10 ml of seedsolution per liter of medium. The fermentation is carried out at atemperature of 28±1° C. and an oxygen concentration of 0.4 mol/m³ in aclean environment where there were no sources of electromagnetic fields.After about 48 hours of fermentation, the concentration of yeast cellsreached about 2×10¹⁰ cells/ml

[0267] The production of ATP-producing yeast is carried out in afermentation process using as seed the activated yeast strain AS2.536 asdescribed in Section 5 7. A schematic of the fermentation process isillustrated in FIG. 6. The fermentation medium is prepared according toa ratio of 2.5 liters of clean water per kilogram of starch and 10kilograms of starch per metric ton of biological fertilizer. Thefermentation medium is inoculated according to a ratio of 10 ml of seedsolution per liter of medium. The fermentation is carried out at atemperature of 28±1° C. and an oxygen concentration of 0.4 mol/m³ forabout 56 hours in a clean environment there were no sources ofelectromagnetic fields. After fermentation, the cell counts reachedabout 2×10¹⁰ cells/ml.

[0268] The mixture of raw materials was prepared according to Table 15and the procedure in Section 5.14.5. TABLE 15 Ratio of raw materialsMaterial Percentage Requirement Dried sludge in powder 80.3% ≧ 150 mesh,water content ≦ form 5% Starch   15% regular starch powder, watercontent ≦ 8%

[0269] The yeast mixture was prepared according to Table 16 and theprocedure described in Section 5.14.5. TABLE 16 Ratio of yeasts (for 1metric ton of fertilizer) Percentage Yeast Quantity (dry weight) NoteNitrogen-fixing yeast 2.0 kg 0.2% Dry yeast AS2.628 powderPhosphorus-decomposing 2.0 kg 0.2% Dry yeast yeast AS2.399 powderPotassium-decomposing yeast 2.0 kg 0.2% Dry yeast AS2.631 powderCarbon-decomposing yeast 2.0 kg 0.2% Dry yeast AS2.982 powderPathogen-suppressing yeast 1.0-2.0 kg 0.1-0.2% Dry yeast IFFI1301 powderChemical-degrading yeast 1.0-2.0 kg 0.1-0.2% Dry yeast IFFI1291 powderOdor-reducing yeast 1.0-2.0 kg 0.1-0.2% Dry yeast IFFI1202 powder Growthfactor producing yeast 25 L 1% Yeast AS2.413 fermentation broth ATPproducing yeast AS2.536 75 L 3% Yeast fermentation broth

[0270] The biological fertilizer was produced by mixing the yeastmixture, sludge, and any optional inorganic materials at a ratioaccording to Table 17. The mixed yeasts and sludge were conveyed to agranulizer to form granules. The granules of the fertilizer were thendried in a two stage drying process. During the first drying stage, thefertilizer was dried in a first dryer at a temperature not exceeding60±2° C. for a period of 5 minutes so that yeast cells quickly becamedormant. The fertilizer was then sent to a second dryer and dried at atemperature not exceeding 65±2° C. for a period of 8 minutes to furtherremove water. The fertilizer was then cool to room temperature. Thefertilizer was then sent to a bulk bag filler for packing. TABLE 17Fertilizer composition (for 1 metric ton of fertilizer) Percentage (dryQuantity weight) Note Raw material mixture 949 kg 94.9% Dry weight Yeastmixture 100 L  4.8% Dry weight

[0271] The present invention is not to be limited in scope by thespecific embodiments described which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A biological fertilizer composition comprising:(I) sludge; (II) at least one of the following: (a) a first yeast cellcomponent comprising a first plurality of yeast cells that fix nitrogen;(b) a second yeast cell component comprising a second plurality of yeastcells that decompose phosphorus compounds; or (c) a third yeast cellcomponent comprising a third plurality of yeast cells that decomposepotassium compounds; and (III) at least one of the following: (d) afourth) yeast cell component comprising a fourth plurality of yeastcells that suppress the growth of pathogenic microorganisms; (e) a fifthyeast cell component comprising a fifth plurality of Yeast cells thatdegrade antibiotics; or (f) a sixth yeast cell component comprising asixth plurality of yeast cells that reduce the odor of the biologicalfertilizer composition.
 2. The biological fertilizer composition ofclaim 1, further comprising at least one of the following: (g) a seventhyeast cell component comprising a seventh plurality of yeast cells thatconvert complex carbon compounds to simple carbohydrates; (h) an eighthyeast cell component comprising an eighth plurality of yeast cells thatoverproduce growth factors; or (i) a ninth yeast cell componentcomprising a ninth plurality of yeast cells that overproduce adenosinetriphosphate.
 3. A biological fertilizer composition comprising: (I)sludge; (II) at least one of the following: (a) a first yeast cellcomponent prepared by culturing a first plurality of yeast cells in afirst electromagnetic field having a frequency in the range of 840 to916 MHz and a field strength of 10 to 200 mV/cm; (b) a second yeast cellcomponent prepared by culturing a second plurality of yeast cells in asecond electromagnetic field having a frequency in the range of 300 to500 MHz and a field strength of 10 to 300 mV/cm; (c) a third yeast cellcomponent prepared by culturing a third plurality of yeast cells in athird electromagnetic field having a frequency in the range of 190 to285 MHz and a field strength of 10 to 200 mV/cm; and (III) at least oneof the following: (d) a fourth yeast cell component prepared byculturing a fourth plurality of yeast cells in a fourth electromagneticfield having a frequency in the range of 30 to 50 MHz and a fieldstrength of 20 to 200 mV/cm: (e) a fifth yeast cell component preparedby comprising a fifth plurality of yeast cells in a fifthelectromagnetic field having a frequency in the range of 70 to 100 MHzand a field strength in the range of 40 to 250 mV/cm; and (f) a sixthyeast cell component prepared by culturing a sixth plurality of yeastcells in a sixth electromagnetic field having a frequency in the rangeof 2160 to 2250 MHz and 2280 to 2380 MHz and a field strength in therange of 100 to 300 mV/cm.
 4. The biological fertilizer composition ofclaim 3, further comprising at least one of the following: (g) a seventhyeast cell component prepared by culturing a seventh plurality of yeastcells in a fourth electromagnetic field having a frequency in the rangeof to 1050 to 1160 MHz and a field strength of 10 to 200 mV/cm; (h) aneighth yeast cell component prepared by culturing an eighth plurality ofyeast cells in an eighth electromagnetic field having a frequency in therange of 1340 to 1440 MHz and a field strength of 20 to 200 mV/cm; and(i) a ninth yeast cell component prepared by culturing a ninth pluralityof yeast cells in a ninth electromagnetic field having a frequency inthe range of 1630 to 1730 MHz and a field strength of 20 to 200 mV/cm.5. The biological fertilizer composition of claim 2 or 4 wherein eachyeast cell component comprises yeast cells that is from the genus ofSaccharomyces.
 6. The biological fertilizer composition of claim 2 or 4wherein each yeast cell component separately comprises cells of aspecies of yeast selected from the group consisting of Saccharomycescerevisiae, Saccharomyces chevalieri, Saccharomyces delbrueekii,Saccharomyces exiguus, Saccharomyces fermentati, Saccharomyces logos,Saccharomyces mellis, Saccharomyces microellipsoides, Saccharomycesoviformis, Saccharomyces rosei, Saccharomyces rouxii, Saccharomycessake, Saccharomyces uvarum Beijer, Saccharomyces willianus,Saccharomyces ludwigii, Saccharomyces sinenses, and Saccharomycescarlsbergensis.
 7. The biological fertilizer composition of claim 2 or 4wherein each yeast cell component separately comprises cells of a strainof yeast selected from the group consisting of Saccharomyces cerevisiaeHansen, ACCC2034, ACCC2035, ACCC2036, ACCC2037, ACCC2038, ACCC2039,ACCC2040, ACCC2041, ACCC2042, AS2.1, AS2.4, AS2.11, AS2.14, AS2.16,AS2.56, AS2.69, AS2.70, AS2.93, AS2.98, AS2.101, AS2.109, AS2.110,AS2.112, AS2.139, AS2.173, AS2.174, AS2.182, AS2.196, AS2.242, AS2.336,AS2.346, AS2.369, AS2.374, AS2.375, AS2.379, AS2.380, AS2.382, AS2.390,AS2.393, AS2.395, AS2.396, AS2.397, AS2.398, AS2.399, AS2.400, AS2.406,AS2.408, AS2.409, AS2.413, AS2.414, AS2.415, AS2.416, AS2.422, AS2.423,AS2.430, AS2.431, AS2.432, AS2.451, AS2.452, AS2.453, AS2.458, AS2.460,AS2.463, AS2.467, AS2.486, AS2.501, AS2.502, AS2.503, AS2.504, AS2.516,AS2.535, AS2.536, AS2.558, AS2.560, AS2.561, AS2.562, AS2.576, AS2.593,AS2.594, AS2.614, AS2.620, AS2.628, AS2.631, AS2.666, AS2.982, AS2.1190,AS2.1364, AS2.1396, IFFI 1001, IFFI 1002, IFFI 1005, IFFI 1006, IFFI1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI 1021, IFFI 1027, IFFI 1037,IFFI 1042, IFFI 1043, IFFI 1045, IFFI 1048, IFFI 1049, IFFI 1050, IFFI1052, IFFI 1059, IFFI 1060, IFFI 1063, IFFI 1202, IFFI 1203, IFFI 1206,IFFI 1209, IFFI 1210, IFFI 1211, IFFI 1212, IFFI 1213, IFFI 1215, IFFI1220, IFFI 1221, IFFI 1224, IFFI 1247, IFFI 1248, IFFI 1251, IFFI 1270,IFFI 1277, IFFI 1287, IFFI 1289, IFFI 1290, IFFI 1291, IFFI 1291, IFFI1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 1301, IFFI 1302, IFFI 1307,IFFI 1308, IFFI 1309, IFFI 1310, IFFI 1311, IFFI 1331, IFFI 1335, IFFI1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI 1340, IFFI 1345, IFFI 1348,IFFI 1396, IFFI 1397, IFFI 1399, IFFI 1411, IFFI 1413; Saccharomycescerevisiae Hansen Var. ellipsoideus (Hansen) Dekker, ACCC2043, AS2.2,AS2.3, AS2.8, AS2.53, AS2.163, AS2.168, AS2.483, AS2.541, AS2.559,AS2.606, AS2.607, AS2.611, AS2.612; Saccharomyces chevalieriGuillermond, AS2.131, AS2.213; Saccharomyces delbrueckii, AS2.285;Saccharomyces delbrueckii Lindner var. mongolicus Lodder et van Rij,AS2.209, AS2.1157; Saccharomyces exiguus Hansen, AS2.349, AS2.1158;Saccharomyces fermentati (Saito) Lodder et van Rij, AS2.286, AS2.343;Saccharomyces logos van laer et Denamur ex Jorgensen, AS2.156, AS2.327,AS2.335; Saccharomyces mellis Lodder et Kreger Van Rij, AS2.195;Saccharomyces microellipsoides Osterwalder, AS2.699; Saccharomycesovijormis Osterwalder. AS2.100; Saccharomyces rosei (Gailliermond)Lodder et kreger van Rij, AS2.287; Saccharomyces rouxii Boutroux,AS2.178. AS2.180, AS2.370. AS2.371; Saccharomyces sake Yabe, ACCC2045;Saccharomyces carlsbergenis Hansen. ACCC2032, ACCC2033, AS2.113 AS2.116AS2.118, AS2.121, AS2.132, AS2.162, AS2.189, AS2.200, AS2.216, AS2.265,AS2.377, AS2.417, AS2.420, AS2.440, AS2.441, AS2.443, AS2.444, AS2.459,AS2.595, AS2.605, AS2.638, AS2.742, AS2.745, AS2.748, AS2.1042;Saccharomyces uvarum Beijer IFFI 1023, IFFI 1032, IFFI 1036, IFFI 1044,IFFI 1072, IFFI 1205, IFFI 1207; Saccharomyces willianus Saccardo,AS2.5, AS2.7, AS2.119, AS2.152, AS2.293, AS2.381, AS2.392, AS2.434,AS2.614, AS2.1189; Saccharomyces sp., AS2.311; Saccharomyces ludwigiiHansen. ACCC2044, AS2.243, AS2.508; and Saccharomyces sinenses Yue,AS2.1395.
 8. The biological fertilizer composition of claim 2 or 4wherein each yeast cell component comprises cells of Saccharomycescerevisiae.
 9. The biological fertilizer composition of claim 2 or 4further comprising an inorganic substrate component.
 10. The biologicalfertilizer composition of claim 2 or 4 wherein the inorganic substratecomponent comprises one or more of rock phosphate, apatite, phosphorite,sylvinite, halite, camalitite, or potassium mica.
 11. The biologicalfertilizer composition of claim 2 which comprises yeast cell components(a) through (f) of claim 1, and yeast cell components (g) thorugh (i) ofclaim
 2. 12. The biological fertilizer composition of claim 4 whichcomprises yeast cell components (a) through (f) of claim 3, and yeastcell components (g) thorugh (i) of claim
 4. 13. The biologicalfertilizer composition of claim 2 or 4 wherein yeast cell component (a)comprises cells of the yeast strain Saccharomyces cerevisiae AS2.628;yeast cell component (b) comprises cells of the yeast strainSaccharomyces cerevisiae AS2.628; yeast cell component (c) comprisescells of the yeast strain Saccharomyces cerevisiae AS2.631; yeast cellcomponent (d) comprises cells of one or more of the following yeaststrain Saccharomyces cerevisiae IFFI1037, IFFI1021, IFFI1051, IFFI1331,IFFI1345, or IFFI1211; yeast cell component (e) comprises cells of oneor more of the following, yeast strain Saccharomyces cerevisiaeIFFI1063, IFFI1211 IFFI1340, IFFI 1215, IFFI1213, IFFI1206, IFFI1211IFFI1210, or IFFI1260; Yeast cell component (f) comprises cells of oneor more of the following yeast strain Saccharomyces cerevisiae AS2.559.AS2.423, AS2.612, AS2.53, AS2.541, or AS2.163; yeast cell component (g)comprises cells of the yeast strain Saccharomyces cerevisiae AS2.982;yeast cell component (h) comprises cells of the yeast strainSaccharomyces cerevisiae AS2.413 and yeast cell component (i) comprisescells of the yeast strain Saccharomyces cerevisiae AS2.536.
 14. Thebiological fertilizer composition of claim 11 or 12 wherein yeast cellcomponent (a) comprises cells of the yeast strain Saccharomycescerevisiae AS2.628; yeast cell component (b) comprises cells of theyeast strain Saccharomyces cerevisiae AS2.628; yeast cell component (c)comprises cells of the yeast strain Saccharomyces cerevisiae AS2.631;yeast cell component (d) comprises cells of one or more of the followingyeast strain Saccharomyces cerevisiae IFFI1037, IFFI1021, IFFI1051, IFFI1331, IFFI1345, or IFFI1211; yeast cell component (e) comprises cells ofone or more of the following yeast strain Saccharomyces cerevisiaeIFFI1063, IFFI1211, IFFI1340, IFFI1215, IFFI1213, IFFI1206, IFFI1211,IFFI1210, or IFFI1260; yeast cell component (f) comprises cells of oneor more of the following yeast strain Saccharomyces cerevisiae AS2.559,AS2.423, AS2.612, AS2.53, AS2.541, or AS2.163; yeast cell component (g)comprises cells of the yeast strain Saccharomyces cerevisiae AS2.982;yeast cell component (h) comprises cells of the yeast strainSaccharomyces cerevisiae AS2.413; and yeast cell component (i) comprisescells of the yeast strain Saccharomyces cerevisiae AS2.536.
 15. Thebiological fertilizer composition of claim 2 or 4, wherein thepluralities of yeast cells are dried.
 16. A method of producing thebiological fertilizer composition of claim 1 or 3, comprising in theorder stated: (I) preparing a mixture of yeast cells by mixing at leastone yeast cell component (a), (b), or (c); with at least one yeast cellcomponent (d), (e), or (f); and (II) adding sludge to said mixture ofyeast cells to form the biological fertilizer composition.
 17. A methodof producing the biological fertilizer composition of claim 2 or 4,comprising in the order stated: (1) preparing a mixture of yeast cellsby mixing at least one yeast cell component (a), (b), or (c): at leastone yeast cell component (d), (e), or (f); with at least one yeast cellcomponent (g), (h), or (i); and (II) adding sludge to said mixture ofyeast cells to form the biological fertilizer composition.
 18. Themethod of claim 16, wherein: the first yeast cell component of (a) isprepared by culturing a first plurality of yeast cells in a firstelectromagnetic field or a first series of electromagnetic fields havinga frequency in the range of 840 to 916 MHz and a field strength of 10 to200 mV/cm; the second yeast cell component of (b) prepared by culturinga second plurality of yeast cells in a second electromagnetic field or asecond series of electromagnetic fields having a frequency in the rangeof 300 to 500 MHz and a field strength of 10 to 300 mV/cm; the thirdyeast cell component of (c) is prepared by culturing a third pluralityof yeast cells in a third electromagnetic field or a third series ofelectromagnetic fields having a frequency in the range of 190 to 285 MHzand a field strength of 10 to 200 mV/cm; the fourth yeast cell componentof (d) is prepared by culturing a fourth plurality of yeast cells in afourth electromagnetic field or a fourth series of electromagneticfields having a frequency in the range of 30 to 50 MHz and a fieldstrength of 20 to 200 mV/cm; the fifth yeast cell component of (e) isprepared by culturing a fifth plurality of yeast cells in a fifthelectromagnetic field or a fifth series of electromagnetic fields havinga frequency in the range of 70 to 100 MHz and a field strength in therange of 40 to 250 mV/cm; and the sixth yeast cell component of (f) isprepared by culturing a sixth plurality of yeast cells in a sixthelectromagnetic field or a sixth series of electromagnetic fields havinga frequency in the range of 2160 to 2250 MHz and 2280 to 2380 MHz and afield strength in the range of 100 to 300 mV/cm.
 19. The method of claim17, wherein the seventh yeast cell component of (g) is prepared byculturing a seventh plurality of yeast cells in a seventhelectromagnetic field or a seventh series of electromagnetic fieldshaving a frequency in the range of 1050 to 1160 MHz and a field strengthof 10 to 200 mV/cm; the eighth yeast cell component of (h) is preparedby culturing an eighth plurality of yeast cells in an eighthelectromagnetic field or a eighth series of electromagnetic fieldshaving a frequency in the range of 1340 to 1440 MHz and a field strengthof 20 to 200 mV/cm; and the ninth yeast cell component of (i) isprepared by culturing a ninth plurality of yeast cells in a ninthelectromagnetic field or a ninth series of electromagnetic fields havinga frequency in the range of 1630 to 1730 MHz and a field strength of 20to 200 mV/cm.
 20. The method of claim 16, wherein said mixture of yeastcells in step (II) is added to sludge, starch, and an inorganicsubstrate component.
 21. The method of claim 17, wherein said mixture ofyeast cells in step (II) is added to sludge, starch, and an inorganicsubstrate component.
 22. The method of claim 16, further comprising inthe order stated: (III) drying said biological fertilizer composition ata temperature not exceeding 65° C. for a period such that the yeastcells become dormant; (IV) drying said biological fertilizer compositionat a temperature not exceeding 70° C. for a period such that the watercontent is less than 5%; (V) cooling said biological fertilizercomposition to ambient temperature; and (VI) forming granules of saidbiological fertilizer composition.
 23. The method of claim 17, furthercomprising in the order stated: (III) drying said biological fertilizercomposition at a temperature not exceeding 65° C. for a period such thatthe yeast cells become dormant; (IV) drying said biological fertilizercomposition at a temperature not exceeding 70° C. for a period such thatthe water content is less than 5%: (V) cooling said biologicalfertilizer composition to ambient temperature; and (VI) forming granulesof said biological fertilizer composition.
 24. A method for enhancingplant growth comprising growing the plant in the presence of abiological fertilizer composition of claim 2 or
 4. 25. The method ofclaim 23 wherein the biological fertilizer composition is applied tosoil at the depth of the major root system of the plant.
 26. The methodof claim 23 wherein about 600 to 1350 kg/ha of the biological fertilizercomposition is used.
 27. The method of claim 23 wherein the plant is acereal crop, vegetable crop, fruit crop, flower crop, or grass crops.28. The method of claim 23 wherein the plant is wheat, barley, corn,soybean, rice, oat, potato, apple, orange, tomato, melon, cherry, lemon,lettuce, carrot, sugar cane, tobacco, or cotton.